DNA vectors containing mutated HIV proviruses

ABSTRACT

The present invention pertains to mutated, non-infectious HIV viral particles, vectors for production of such particles and vaccines employing such vectors. The non-infectious particles are obtained by introducing a number of inactivating mutations into a native viral genome. These mutations are designed so as to minimize the probability of genetic reversion to an infectious virus, while retaining the basic protein content and immunogenic properties of a wild-type virion. The altered viral genome expresses proteins that can assemble into non-infectious particles which contain immunogenic components of the virus, but which are unable to infect cells. The preferred mutations are introduced in at least one amino acid position of the nucleocapsid (NC) protein in combination with at least one other mutation in an amino acid position of the reverse transcriptase (RT) protein or the In protein. In one embodiment, the mutations to the native HIV genome may also be made in at least one amino acid position of the NC protein, at least one position in the RT protein, and at least one position in the integrase (In) protein. In another embodiment, the mutations to the native HIV genome may be introduced in clusters, where two or more mutations are made in the NC protein, the RT protein, the In protein, or any combinations thereof.

RELATED CASE INFORMATION

The present invention claims priority to U.S. Provisional ApplicationNo. 60/242,589, filed Oct. 23, 2000 entitled: DNA Vectors ContainingMutated HIV Proviruses and U.S. Provisional Application No. 60/253,432,filed Nov. 28, 2000 entitled: DNA Vectors Containing Mutated HIVProviruses.

GOVERNMENT SUPPORT

Work described herein was supported by one or more of Grants Nos.AI41365, AI34757 AI-85343 and RR00168 from the National Institutes ofHealth and Contract No. NO1-CO-56000 awarded by the National CancerInstitute of NIH. The U.S. Government has certain rights in theinventions pursuant to this funding.

BACKGROUND OF THE INVENTION

The technical field of the invention is molecular biology and, inparticular, vectors producing non-infectious particles that can be usedto induce viral specific immune system responses.

Human immunodeficiency virus (HIV), the virus which causes acquiredimmune deficiency syndrome (AIDS), is a member of the retrovirus family.In particular, the HIV virus belongs to the lentivirus subfamily ofretroviruses. The HIV virus contains two strands of single-strandedgenomic ribonucleic acid (RNA) associated with two molecules of reversetranscriptase, an enzyme that catalyzes the process of “reversetranscription” to transcribe genomic RNA into double-stranded DNA. HIValso contains other nucleoid proteins, such as a protease enzyme and anintegrase enzyme. The HIV genome, including the nucleoid proteins, issurrounded by a viral coat, known as the nucleocapsid, which consists oftwo layers of proteins. The HIV genome is further surrounded by an outerenvelope coat, which is derived from the membrane of the host-cell.

The single-stranded RNA of the HIV genome encodes three differentcategories of proteins: the structural proteins encoded by the gag, poland env genes; the regulatory proteins encoded by the tat and rev genes;and the accessory proteins encoded by the vpu, vpr, vif and nef genes.The HIV genome also has a repeated sequence, known as the long terminalrepeat (LTR), at both the 5′ and the 3′ end of the genome. The 5′ LTRcontains enhancer and promoter sequences that are necessary for viraltranscription, while the 3′ LTR sequence is required for polyadenylatingthe transcripts that are created from the RNA genome. (Kuby, J.,Immunology, 3^(rd) ed. (W. H Freeman and Company (1997)).

Numerous studies have investigated the role of these different proteins,in particular, the role of the structural proteins encoded by the gagand pol genes. To date, attempts have focused on separately mutatingproteins such as the reverse transcriptase (RT), integrase (In) andnucleocapsid (NC) in order to characterize and modify their function.(See e.g. Kim, et al., J. Biol. Chem., 271:4872–4878 (1996); Winters etal., J. Virol. 74:10707–10713 (2000); Lins et al., Biophys. J.76:2999–3011 (1999); Ellison et al., J. Biol. Chem. 270:3320–3326(1995); Zheng et al., Proc. Natl. Acad. Sci. 93:13659–13664 (1996);Leavitt et al., J. Virol. 70:721–728 (1996); Tanchou et al., J. Virol.72:4442–4447 (1998); Druillennec et al., J. Biol. Chem. 274:11283–11288(1999); and Schwartz et al., J. Virol. 71:9295–9305 (1997)).

In addition, attempts have been made to produce non-infectious virusparticles, in particular by targeting the nucleocapsid protein. (SeeAldovini et al., J. Virol. 64:1920–1926 (1990) and Poon et al., J.Virol. 70:6607–6616 (1996)). In U.S. Pat. No. 5,919,458, Aldovini etal., describes one approach to the construction of non-infectious HIVparticles. This approach involves generating nucleotide alterations inthe cis-acting RNA packaging site, also known as the Ψ site, and in thecysteine rich carboxy-terminal region of the gag gene, to create HIVmutants that were defective for RNA packaging. Another approach toconstructing non-infectious HIV particles is described by Poon et al.,J. Virol. 70:6607–6616, (1996). In this study, non-infectious particleswere created by altering the nucleocapsid structure to preclude viralRNA incorporation. However, these studies relied on generating deletionmutants of the NC domain in which entire structural domains of the NCwere removed in order to prevent recombination to wild-type HIV. As aconsequence, the alteration in the protein structure of the HIV virusprevents the virion from acting as a suitable antigen to elicit animmune response. Furthermore, there still remained the concern forreversion to wild-type virus and the generation of infectious particles.

There is also an interest in the development of vaccines to HIV toprotect against, or at least retard the progression of, AIDS. Thepotential efficacy of such vaccines has been suggested by studies in thesimian AIDS model systems and in limited human trials. Challenging thepatient with either an attenuated or inactivated whole virion conferssome immunity. Studies have shown that vaccines composed of whole,inactivated virions of simian immunodeficiency virus (SIV) confer atleast partial protection against challenge with live virus. (See eg.,Langlois et al., Secience 255:292–293 (1992); Le Grand et al., Nature355:684 (1992); Osterhaus et al., ibid., pp. 684–685; Cranage et al.,ibid., pp. 685–686).

Production of inactivated HIV vaccines involves physical and chemicalinactivation treatments necessary to render a non-infectious particle.However, such treatments can result in loss of immunogenicity due topartial destruction of the virions, thereby limiting the effectivenessof the immune response. A method that leaves virion structures intact,yet renders the virions non-infectious, would be a significantimprovement in vaccine development.

One approach is the use of a DNA vaccine where the subject is inoculatedwith DNA molecules carrying a gene that encodes for a defective virion.DNA vaccines have a number of potential advantages over moreconventional vaccine formulations. For example, multiple antigens can beexpressed from a single DNA construct. In addition, because DNA vaccinesexpress antigens in their native form, both humoral and cellularresponses from the immune system are expected to be observed. Ofparticular importance is the generation of mucosal immunity because themajor entry route for these retroviruses is typically through anorifice, which is lined with mucosa.

Accordingly, a need exists for producing a non-infectious virion whichretains structural integrity in order to elicit an immune response,while preventing reversion to the wild-type virus. A need also existsfor a DNA vaccine comprising a non-infectious virions capable ofeliciting appropriate immune response in a subject without the risk ofcausing infection. A need also exists for DNA vaccines that can bespecifically targeted to cells lining the passageways of the body.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that severalcombinations of point mutations to the wild-type HIV genome can beemployed to create composite safeguards in the production ofnon-infectious HIV particles and reduce or eliminate reversion towild-type virus. The mutations also ensure that the non-infectiousparticles retain the ability to elicit an immune response in a subject.The mutations taught herein target various steps in the HIV replicationcycle to produce non-infectious HIV particles. The steps of the HIVreplication cycle typically include the binding of a mature HIV virionto the host cell membrane, the reverse transcription of the viral RNA tocreate double-stranded DNA and the incorporation of this double-strandedDNA into the genome of the host cell. Once the viral genome has beenintegrated into the host cell genome, the host cell's own cell machinerytranscribes the HIV genes along with the host cell's own genes, therebyproducing the viral proteins that will assemble into mature, infectiousHIV particles. The invention targets each of these independent steps toprovide a construct with mutations in proteins associated with eachstep, resulting in a non-infectious particle that retains the necessarystructural features required to elicit the desired immune response, yetis unable to revert to a wild-type virus and cause infection. Thesemutations are designed to create a composite safety net, diminishing thepossibility of generating an infectious particle, while retaining thebasic protein content and immunogenic properties of a wild-type HIVvirion.

Because replication of the HIV virus requires a mature virion torecognize and bind to the target host cell, mutating the NC protein soas to prevent the viral RNA from packaging into a mature genome willpreclude the production of a mature virion. If the viral RNA were topackage and form a mature genome, mutations in the RT protein thatprevent the reverse transcription and the synthesis of double-strandedDNA would produce a HIV particle that is incapable of integrating intothe genome of the host cell, and thus, non-infectious. If adouble-stranded DNA copy of the viral genome were produced, mutations inthe In protein that would prevent viral DNA from infiltrating the genomeof a host cell would prevent the HIV genome from being transcribed andtranslated by the host cell's own cell mechanisms, thereby rendering theparticle non-infectious. Thus, the novel combination of these mutationsyields numerous safety precautions that will ensure the production ofnon-infectious, non-replicating HIV particles.

The present invention pertains to a nucleic acid construct capable ofproducing human immunodeficiency virus particles that can be used toelicit immune responses without causing viral infection. In particular,these mutations are made in one or more distinct regions of the viralgenome, such as the NC encoding region, the RT encoding region and theIn encoding region. These mutations are designed to alter thefunctionality of the nucleocapsid protein, the reverse transcriptaseprotein and the integrase protein, while preserving the tertiary (orquarternary) structure of each protein. The mutated HIV genome expressesmutant NC, RT and In proteins that can assemble into non-infectiousparticles which contain immunogenic components of the virus, but whichare unable to infect cells. These mutations allow the production ofnon-infectious, immunogenic particles and provide a means for obtainingvaccines and other diagnostic reagents based on particles that areimmunogenic, but not infectious.

In one aspect of the present invention, two or more mutations are madein a wild-type HIV genome. The preferred mutations are introduced in atleast one amino acid position of the NC protein in combination with atleast one other mutation in an amino acid position of the RT protein orthe In protein. In one embodiment, the mutations to the native HIVgenome may also be made in at least one amino acid position of the NCprotein, at least one position in the RT protein, and at least oneposition in the In protein. In another embodiment, the mutations to thenative HIV genome may be introduced in clusters, where two or moremutations are made in the NC protein, the RT protein, the In protein, orany combinations thereof.

In another aspect of the present invention, the mutated HIV construct istransfected into a mammalian cell line to produce mutant,non-infectious, non-replicating HIV particles.

In yet another aspect, the mutated HIV construct is used as a vaccine togenerate an immune response, eg. a mucosal or systemic immune responsein a subject. These non-infectious viral particles provide analternative and advantageous method for the preparation of a whole virusvaccines. Such vaccines can be used to induce an anti-HIV response in anindividual, either prior to or after infection with HIV, resulting inenhanced resistance by the individual to the virus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a photograph of a Western blot of viral particle productionby SIV mutated constructs upon transfection into 293T cells. The blotwas probed with a macaque SIV polyclonal serum that reacts predominantlywith the SIV Env products.

FIG. 1B is a photograph of a Western blot of viral particle productionby SIV mutated constructs upon transfection into 293T cells. The blotwas probed with a macaque SIV polyclonal serum that reacts predominantlywith the SIV Gag products.

FIG. 1C is a photograph of an agarose gel displaying RT-PCR analysis ofthe genomic RNA content of SIV mutated particles. RNA amplification wascarried out with SIV gag-related primers on viral RNA extracted frompelleted virions. Lanes a and b are respectively RT-PCR and PCRreactions carried out on the RNA samples.

FIG. 1D is a photograph of an agarose gel displaying RT-PCR analysis oftotal viral RNA accumulated intracellularly 48 hours after transfectionwith SIV constructs. Nucleic acid amplification was carried out with SIVtat-related primers on total cellular RNA extracted from transfected239T cells. Lanes a and b are RT-PCR (a) and PCR (b) reactions carriedout on the total cellular RNA samples.

FIG. 2 is a graph depicting SIV viral loads in macaques challengedrectally by SIVmac239 (serum RT-PCR). Each time point represents theaverage and standard error of the values of viral loads detected in thethree animals of one regimen group and reported in Table 10.

DETAILED DESCRIPTION

So that the invention is more clearly understood, the following termsare defined:

The term “HIV,” as used herein, refers to all strains and permutationsof HIV. The term HIV can include, but is not limited to HIV-1 and HIV-2.

The term “mutation,” as used herein, refers to any alteration of the gagand/or pol gene that inactivates the functionality of the proteinproduced by that gene. Such mutations can include, but are not limitedto, an amino acid substitution wherein a native amino acid is replacedwith an alanine or other biologically comparable amino acid residue,including, but not limited to glycine, valine, and leucine, or adeletion of any portion of the gag and/or pol gene.

The term “portion” or “fragment” as used herein refers to an amino acidsequence of the gag or pol genes that has fewer amino acids than theentire sequence of the gag and/or pol genes.

The term “cluster” or “cluster of mutations” as used herein refers toany mutations made in two or more residues that are located withinthree, five, seven, nine or eleven amino acid positions of each other.Preferably, cluster refers to two or more mutations within seven aminoacids. Cluster can also refer to mutations made within 1 amino acidresidue upstream or downstream of the site-specific mutation or within 2amino acid residues upstream or downstream of the site-specificmutation.

The term “coding sequence” or a sequence which “encodes” or sequence“encoding” a particular protein, as used herein refers to a nucleic acidmolecule which is transcribed (in the case of DNA) and translated (inthe case of messenger mRNA) into a polypeptide in vitro or in vivo whenplaced under the control of appropriate regulatory sequences

The terms “5′”, “3′”, “upstream” or “downstream” are art-recognizedterms that describe the relative position of nucleotide sequences in aparticular nucleic acid molecule relative to another sequence.

The term “promoter” is used herein refers to the art recognized use ofthe term of a nucleotide region comprising a regulatory sequence,wherein the regulatory sequence is derived from a gene which is capableof binding RNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

The term “regulatory sequence” is art-recognized and intended to includecontrol elements such as promoters, enhancers and other expressioncontrol elements (e.g., polyadenylation signals), transcriptiontermination sequences, upstream regulatory domains, origins ofreplication, internal ribosome entry sites (“IRES”), enhancers, enhancersequences, post-regulatory sequences and the like, which collectivelyprovide for the replication, transcription and translation of a codingsequence in a recipient cell. Not all of these regulatory sequences needalways be present so long as the selected coding sequence is capable ofbeing replicated, transcribed and translated in an appropriate hostcell. Such regulatory sequences are known to those skilled in the artand are described in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990), the teachingsof which are herein incorporated in their entirety by reference. Itshould be understood that the design of the viral vector may depend onsuch factors as the choice of the host cell to be transfected and/or theamount of protein to be expressed.

The term “operably linked” as used herein refers to an arrangement ofelements wherein the components are configured so as to perform theirusual function. Thus, control elements operably linked to a codingsequence are capable of effecting the expression of the coding sequence.The control elements need not be contiguous with the coding sequence, solong as they function to direct the expression of the coding sequence.For example, intervening untranslated yet transcribed can be presentbetween a promoter sequence and the coding sequence and the promotersequence can still be considered “operably linked” to the codingsequence.

The term “transfection” is used herein refers to the uptake of anexogenous nucleic acid molecule by a cell. A cell has been “transfected”when exogenous nucleic acid has been introduced inside the cellmembrane. A number of transfection techniques are generally known in theart. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al.(1989) Molecular Cloning, a laboratory manual, Cold Spring HarborLaboratories, New York, Davis et al. (1986) Basic Methods in MolecularBiology, Elsevier, and Chu et al. (1981) Gene 13:197, the teachings ofwhich are herein incorporated in their entirety by reference. Suchtechniques can be used to introduce one or more exogenous nucleic acidmolecules into suitable host cells. The term refers to both stable andtransient uptake of the nucleic acid molecule.

The term “gene transfer” or “gene delivery” as used herein refers tomethods or systems for reliably inserting foreign DNA into host cells.Such methods can result in transient expression of non-integratedtransferred DNA, extra-chromosomal replication and expression oftransferred replicons (e.g., episomes), or integration of transferredgenetic material into the genomic DNA of host cells. Gene transferprovides a unique approach for the treatment of acquired and inheriteddiseases. A number of systems have been developed for gene transfer intomammalian cells. (See, e.g., U.S. Pat. No. 5,399,346, the teachings ofwhich are herein incorporated in their entirety by reference).

The term “subject” as used herein refers to any living organism in whichan immune response is elicited. The term subject includes, but is notlimited to, humans, nonhuman primates such as chimpanzees and other apesand monkey species; farm animals such as cattle, sheep, pigs, goats andhorses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

The terms “polypeptide” and “protein” are used interchangeably hereinand refer to a polymer of amino acids and includes full-length proteinsand fragments thereof. As will be appreciated by those skilled in theart, the invention also includes nucleic acids that encode thosepolypeptides having slight variations in amino acid sequences or otherproperties from a known amino acid sequence. Amino acid substitutionscan be selected by known parameters to be neutral and can be introducedinto the nucleic acid sequence encoding it by standard methods such asinduced point, deletion, insertion and substitution mutants. Minorchanges in amino acid sequence are generally preferred, such asconservative amino acid replacements, small internal deletions orinsertions, and additions or deletions at the ends of the molecules.These modifications can result in changes in the amino acid sequence,provide silent mutations, modify a restriction site, or provide otherspecific mutations. Additionally, they can result in a beneficial changeto the encoded protein.

The term “homology” or “identity” as used herein refers to thepercentage of likeness between nucleic acid molecules or proteinmolecules, including codon-optimized nucleic acid molecules. Todetermine the homology or percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, even more preferably at least 60%,and even more preferably at least 70%, 80%, or 90% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two amino acidsequences can be determined using the Needleman and Wunsch ((1970) J.Mol. Biol. (48):444–453, the teachings of which are herein incorporatedin their entirety by reference) algorithm which has been incorporatedinto the GAP program in the GCG software package, using either a Blossom62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another example, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package, using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of1, 2, 3, 4, 5, or 6. In yet another example, the percent identitybetween two amino acid or nucleotide sequences is determined using thealgorithm of E. Meyers and W. Miller (CABIOS, 4:11–17 (1989)) which hasbeen incorporated into the ALIGN program (version 2.0), using a PAM120weight residue table, a gap length penalty of 12 and a gap penalty.

The present invention pertains to the introduction of a number ofinactivating mutations into a viral genome of an immunodeficiency virus.The viral genome can be that of HIV. All strains and permutations of HIVare within the scope of this invention, for example HIV-1 and HIV-2. Theaccession numbers of HIV-1 and HIV-2 are NC001802 and NC001722,respectively, and are available from the NCBI database. Moreover,molecular clones of the HIV virus, including, but not limited to, HXB2are within the scope of this invention. The nucleotide sequence of HXB2is shown in SEQ ID NO: 1, and the amino acid sequence of HXB2 is shownin SEQ ID NO: 2. Different genetic regions within the HXB2 that areparticularly important in the present invention include the reverse RTgene, the In gene, and the NC gene. The nucleotide sequence of the RTgene is shown in SEQ ID NO: 3, and the amino acid sequence of RT isshown in SEQ ID NO: 4. The nucleotide sequence of the In gene is shownin SEQ ID NO: 5, and the amino acid sequence of In is shown in SEQ IDNO: 6. The nucleotide sequence of the NC gene is shown in SEQ ID NO: 7,and the amino acid sequence of NC is shown in SEQ ID NO: 8. Thenucleotide sequence of gag gene is SEQ ID NO: 9 and the amino acidsequence is SEQ ID NO: 10. The amino acid sequence of molecular cloneslike HXB2 can be obtained from a database, e.g., NCBI, and the actualclone from the NIH AIDS Research and Reference Reagent Program Catalog.

The mutated viral genome of the invention is used to transfect a hostcell. Once inside the host cell, the viral genome can undergo cellularprocessing, including transcription. The expressed mutated and wild-typeviral proteins are used to assemble a defective (non-infectious) viralparticles (also known as virions). These mutated (or defective) viralparticles have a protein content that is similar to the wild-type (ornative) virion, thus the defective viral particle retains immunogenicproperties similar to wild-type virions.

Further details of the invention are described in the followingsections:

I. The Nucleocapsid, Reverse Transcriptase, and Integrase Proteins ofHIV

The present invention provides a novel combination of mutations to thewild-type HIV genome that create numerous safety precautions to ensurethe production of non-infectious HIV particles. The mutations taughtherein target various steps in the HIV replication cycle that arecatalyzed or controlled by the NC, RT and In proteins. These mutationsites are conservative sites found throughout the various strains ofHIV.

In one embodiment of the instant invention, mutant HIV viruses aredisclosed wherein at least two independent genes are mutated. In thisembodiment, at least one mutation is made in the NC gene and one or moremutations also occurs in the RT gene and/or the In gene. This HIVconstruct is capable of eliciting an immune response in an inoculatedhost, thereby providing immunological protection against the HIV virus.In one aspect of the current invention, immunity is specificallyelicited by the introduction of a mutated HIV construct into differenttissues. In a preferable embodiment, one or more mutations occurs in allthree genes encoding the NC, RT and In proteins. These genes can bemutated by site directed mutagenesis.

Some amino acid residues that appear to be important to the function ofNC, RT and In have been characterized. (See Ellison et al., J. Biol.Chem. 270:3320–3326 (1995); Kim et al., J. Biol. Chem. 271:4872–4878(1996), the teachings of which are incorporated herein in their entiretyby reference). Some of these amino acid residues were chosen as mutationsites because they retain the tertiary (or quaternary) structure of themutated protein so that it resembles the wild-type protein with respectto immunogenic properties. It is not sufficient that the mutated proteinhas immunogenicity itself, it must have the same or similar immunogenicproperties as found in the wild-type protein in order to elicit thedesired immune response. In addition, amino acid residues selected forsite specific mutation were those that would prevent viral RNApackaging, as well as disrupting RT and In function.

Accordingly, the present invention targets regions in the nucleocapsidp7 protein (NCp7), the p66/p51 reverse transcriptase protein (RT) andthe p32 integrase protein (In). The region of the gag gene that encodesthe NCp7 has been targeted, because NCp7 is known to play a significantrole in the creation of mature virus particles. NCp7 has been shown tobe responsible for the packaging of viral RNA in the HIV virus.According to these studies, packaging of retroviral RNA requires aninteraction between the NCp7 domain of the Gag polypeptide precursor andthe RNA packaging site (also referred to as the Ψ-site), located in theHIV genome between the 5′ LTR and the gag initiation codon. (SeeAldovini et al., J. Virol. 64:1920–1926 (1990); Poon et al., J. Virol70: 6607–6616 (1996), the teachings of which are herein incorporated intheir entirety by reference).

For the purposes of the invention, the NCp7 has been divided into fiveregions, which include the 5′ flanking region (amino acids 1–14), thefirst zinc finger domain, also known as the 5′ Cys-His box (residues15–28 of SEQ ID NO: 8), the basic amino acid linking region (residues29–35 of SEQ ID NO: 8), the second zinc finger, also known the 3′Cys-His box (residues 36–49 of SEQ ID NO: 8) and the 3′ flanking region(residues 50–55 of SEQ ID NO: 8). The two, highly conserved Cys-Hisboxes, which take the form of CX₂CX₄HX₄C, are in well-defined spatialproximity, while the N-terminal and C-terminal sequences remainflexible. (Druillennec et al., Proc. Natl. Acad. Sci. 96:4886–4891(1999), the teachings of which are herein incorporated in their entiretyby reference). The NCp7 protein has been found to interact with thereverse transcriptase protein to form a 1:1 complex. These studies haveshown that the 5′ and 3′ Cys-His boxes, and the linking region, arenecessary for reverse transcriptase binding. Alterations in thesedomains prevent the formation of the NCp7-reverse transcriptase complex.(Druillennec et al., J. Biol. Chem. 274:11283–11288 (1999), theteachings of which are herein incorporated in their entirety byreference).

A previous study by the present inventor and others found that mutationsin the NCp7 domain of the gag precursor polypeptide reduced theefficiency of RNA packaging in the HIV virus. (Poon, et al. J. Virol.70:6607–6616 (1996), the teachings of which are incorporated herein intheir entirety by reference). In this study, the highly basic NCp7protein was mutated using an alanine amino acid to replace variouspositively-charged amino acid residues. The positively-charged aminoacid residues of the NCp7 protein include five amino acid residues inthe N-terminal region (arginine 3, arginine 7, arginine 10, lysine 11and lysine 14), three amino acid residues in the 5′ Cys-His box (lysine20, histidine 23 and arginine 26), four amino acid residues in thelinking region (arginine 29, arginine 32, lysine 33 and lysine 34), fouramino acid residues in the 3′ Cys-His box (lysine 38, lysine 41,histidine 44 and lysine 47), and one amino acid residue in theC-terminal region (arginine 52). The twenty-eight mutants studied byPoon et al. included single mutations at each of the basic amino acidpositions, as well as “clusters” of mutations within the regions, andcombinations thereof. Clusters has been defined as mutations in two ormore amino acid residues that are located within three, five, seven,nine or eleven amino acid positions of each other. (See Table 1).

TABLE 1 Mutations in the NCp7 protein M Q R G N F R N Q R K I V K C F NC G K E G H T A R N C R A P R K K G C W K C G K E G H Q M K D C T E R QA N mutated a.a# construct name - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 3 pR3 - - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 7 pR7 - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -10 pR10 - - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -11 pK11 - - - - - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -14 pK14 - - - - - - - - - - - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -20 pK20 - - - - - - - - - - - - - - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 23pH23 - - - - - - - - - - - - - - - - - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 26pR26 - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - 29pR29 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - - - 32pR32 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - - 33pK33 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - 34pK34 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - - - - - - - - - - - - - - - 38pK38 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - - - - - - - - - - - - 41pK41 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - - - - - - - - - 44pH44 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - - - - - - 47pK47 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - 52 pR52 - - - - - - - - - - - - - A - - - - - A - - - - - A - -A - - A A A - - - A - - A - - - - - A - - - - - - - -14-20-26-29-32-33-34-38-41-47 pM1-2/BR - - - - - - - - - - - - -A - - - - - A - - - - - A - - - - - - - - - - - A - - A - - - - -A - - - - - - - - 14-20-26-38-41-47 pM1-2 - - - - - - - - - - - - -A - - - - - A - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 14-20-26p14-20-16 - - - - - - - - - - - - - - - - - - - A - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 20-26p20-26 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - A - - - - - A - - - - - - - - 38-41-47p38-41-47 - - - - - - - - - - - - - - - - - - - - - - - - - - - - A - -A A A - - - - - - - - - - - - - - - - - - - - - 29-32-33-34pBR - - - - - - - - - - - - - - - - - - - - - - - - - - - - A - -A - - - - - - - - - - - - - - - - - - - - - - - 29-33p29-33 - - - - - - - - - AA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - 10-11-52 p10-11-52 - - - - - - - - - AA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -10-11 p10-11 - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - 10-52 p10-52 - - - - - - - - - -A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -A - - - 11-52 p11-52 Schematic representation of alanine scanningmutations within the HIV-1 NC p7 protein. The amino acid sequence ofHIV-1 NC p7 is listed above the alanine substitutions for each mutant.The specific amino acid residue(s) (aa) modified in each mutant and thename of the corresponding plasmid construct are shown on the right.

The results of this study by Poon et al. show that several NCp7 mutantsproduced non-infectious HIV particles. Of the twenty-eight mutantsstudied, eleven were found to reduce or eliminate the infectivity of theresulting HIV mutant particles. (See Table 2, below). The results inTable 2 show two constructs (pR3 and pK14) in which single amino acidsubstitutions resulted in non-infectious particles. These mutantsinclude two single amino acid mutations, one at arginine 3 and the otherat position lysine 14, which immediately precedes the 5′ Cys-His box.The other mutants that yielded non-infectious particles includedclusters of mutations in the 5′ flanking region, the 5′ Cys-His box, inthe basic amino acid linker region, and in the 3′ Cys-His box, as wellas combinations thereof. In addition to the mutations in the NC protein,the present invention combines mutations in the NC protein withmutations in the RT and/or the In proteins to provide additionalsafeguards for preventing reversion to an infectious virus.

TABLE 2 Effects of alanine substitutions in NC p7 on viral genomic RNAincorporation and infectivity* RNA incorporation Construct (% of wt)Infectivity Group pHXB2gpt 100 +++ A pR3  81.63 + 11.46 − C pR7 52.80 +5.22 + B pR10 47.20 + 4.29 + B pK11 24.90 + 3.13 + B pK14 22.30 + 1.67 −C pK38 63.90 + 2.58 + B pK47 59.46 + 1.71 + B pM1-2/BR  0.33 + 0.33 − CpM1-2  1.33 + 0.23 − C p14-20-26 12.56 + 2.51 − C p38-41-47  0.70 + 0.35− C pBR 14.03 + 2.12 − C p10-11-52 10.30 + 3.85 − C p10-11 32.83 + 5.18− C p10-52 48.03 + 3.15 + B p11-52 56.76 + 9.22 + B *Relative nucleicacid content of viral particles was measured by RT-PCR. RT-PCRs usinggag-specific primers were carried out to detect viral genomic RNAincorporation. The average of values + standard error from threeindependent experiments is reported for each mutant. The infectivitygroup to which each mutant was assigned is also listed. wt, wild type.

Accordingly, in addition to the mutations in the NC protein, the presentinvention also targets the portion of the pol gene that encodes theintegrase protein. The integrase enzyme is responsible for incorporatingthe viral DNA into the DNA of the host cell. Once the viral DNA has beeninserted, the host cell's own transcription and translation mechanismscause the cell to produce viral particles.

Formed from a single polypeptide chain, the integrase protein has threefunctional domains. These three domains consist of the N-terminal domain(residues 1–50 of SEQ ID NO: 6), the core region (residues 51–212 of SEQID NO: 6), and the C-terminal domain (residues 213–280 of SEQ ID NO: 6).(See e.g., Lins et al., Biophys. J. 76:2999–3011 (1999); Leavitt et al.J. Virol. 70:721–728 (1996); Ellison et al., J. Biol. Chem.270:3320–3326 (1995); Zheng et al., Proc. Natl. Acad. Sci.93:13659–13664 (1996), the teachings of which are incorporated herein intheir entirety by reference).

The N-terminal domain of integrase contains a pair of highly conservedHis and Cys residues, also known as the HHCC motif, which has been foundto bind zinc with a stoichiometry of one zinc ion per integrase protein.(Zheng et al., Proc. Natl. Acad. Sci., 93:13659–13664 (1996), theteachings of which are herein incorporated in their entirety byreference). Studies have shown that mutations in the HHCC domain affectthe ability of integrase to bind zinc ions, which in turn affects thecatalytic activity of the integrase enzyme. (See e.g., Ellison et al.,J. Biol. Chem. 270:3320–3326 (1995); Zheng et al., Proc. Natl. Acad.Sci., 93:13659–13664 (1996), the teachings of which are hereinincorporated in their entirety by reference). When the integrase proteinbinds a zinc ion, its catalytic reaction rate is between 5- and 15-foldgreater than an integrase enzyme that contains no zinc. (Zheng et al.,Proc. Natl. Acad. Sci., 93:13659–13664 (1996), the teachings of whichare herein incorporated in their entirety by reference).

The core domain of the integrase protein contains the highly conservedamino acid sequence of two aspartic acid residues followed by a glutamicacid residue (commonly referred to as the D,D35E motif), with aconserved spacing of 35 residues between the second and third acidicresidues. These D,D35E residues (aspartic acid 66, aspartic acid 118 andglutamic acid 154) are commonly called the catalytic triad, because theyform the active site of the catalytic domain. (Lins et al. Biophys. J.,76:2999–3011 (1999), the teachings of which are herein incorporated intheir entirety by reference). Mutations at each of these residuesimpairs the ability of the HIV virus to integrate into a host cell andform a provirus. (Leavitt, et al., J. Virol. 70:721–728 (1996), theteachings of which are herein incorporated in their entirety byreference).

The present invention also targets the region of the pol gene thatencodes for the reverse transcriptase protein. Reverse transcriptase isformed by cleaving the Gag-Pol polypeptide precursor to produce ahomodimer of two p66 molecules. Initially, each p66 molecule contains apolymerase and an RNase H domain, but the RNase H domain of one subunitis subsequently removed to create a p66-p51 reverse transcriptaseheterodimer. The polymerase domain of the reverse transcriptase proteinconsists of four subdomains, commonly referred to as the “fingers,”“palm,” “thumb” and “connection” regions. The “fingers” region (residues1–84 and 120–150 of SEQ ID NO: 4) contains mixed β-strands and threeα-helices. In particular, the β3–β4 loop encodes for amino acids 67–78of the reverse transcriptase protein. The five β-strands of the “palm”region (residues 85–119 and 151–243 of SEQ ID NO: 4) interact and formhydrogen bonds with the four β-strands of the “thumb” region (residues244–322 of SEQ ID NO: 4). The “connection” subdomain (residues 323–437of SEQ ID NO: 4), which connects the polymerase and RNase H domains, iscomposed of a large β-sheet and two α-helices. Studies have shown thatthe residues of the “fingers,” “palm” and “thumb” subdomains form themajority of RT-DNA contacts when the enzyme binds to DNA. (Turner etal., J. Mol. Biol. 285:1–32 (1999), the teachings of which are hereinincorporated in their entirety by reference).

While the polymerase subdomains of the p51 and p66 have identical aminoacid sequences, the packaging of the subdomains found within eachmolecule is drastically different. In the p66 molecule, the subdomainsof the polymerase region pack together to form a configuration, commonlyreferred to as an “open-right hand,” in which the three catalyticresidues, aspartic acid 110, aspartic acid 185 and aspartic acid 186,are exposed. The subdomains of the p51 molecule, however, are packagedso that the “fingers” close over the “palm” region and, thus, cover thecatalytic residues. Consequently, p51 is a catalytically inactivemolecule. (Turner et al., J. Mol. Biol. 285:1–32 (1999), the teachingsof which are herein incorporated in their entirety by reference).

Other regions of the HIV genome can also be mutated in combination withthe mutations made in the NC, RT and/or In genes. For example, thenucleotide sequence coding for the long terminal repeats (LTR) can bemutated. (See e.g., U.S. Pat. Nos. 5,912,338, 5,439,809, 5,866,320 &5,889,176, the entire teaching of which is incorporated herein byreference). These viral genes when mutated along with the RT and In geneprovide for additional safety by minimizing the possibility of viralreversion.

Also within the scope of the invention is a construct that comprises anenvelope gene, and variations thereof, in addition to one or moremutations in the NC, In and/or RT genes. The HIV envelope protein hasbeen extensively described, and the amino acid and RNA sequencesencoding HIV envelope from a number of HIV strains are known (Myers etal., 1992. Human Retroviruses and AIDS. A compilation and analysis ofnucleic acid and amino acid sequences. Los Alamos National Laboratory,Los Alamos, N. Mex.). The envelope protein has hypervariable domainsthat have extensive amino acid substitutions, insertions and deletions.Sequence variations in these domains result in up to 30% overallsequence variability between envelope molecules from the various viralisolates.

In one embodiment of this invention, a mutated HIV genome is packagedinto an appropriate plasmid vehicle. A host cell is then transfectedusing this mutated construct. Once the viral construct enters the hostcell, the mutated viral genome can be transcribed and translated leadingto mutated viral proteins that are able to assemble into anon-infectious viral particle.

II. Construction of the DNA Vaccine

The HIV construct comprising mutations in viral genes and regulatoryelements can be used to produce a DNA vaccine. DNA vaccines have anumber of demonstrated and potential advantages over more conventionalvaccine formulations. Stimulation of both humoral and cellular responsesis usually observed, as antigens are expressed in their native form andcorrectly presented to the immune system. These responses can involveMHC class I-restricted cytotoxic T-lymphocytes (CTLs), even in animalmodels that do not normally exhibit strong CTL responses to vaccination.Multiple antigens can also be expressed from a single vaccine construct.A variety of different routes and methods of immunization are availablefor DNA vaccination including subcutaneous, intradermal, intramuscular,nasal, vaginal and mucosal. DNA can be delivered in a liposomeformulation by injection or by gene gun delivery. The DNA can bedelivered in such a way as to generate a systemic response and a mucosalresponse at the same time. The DNA may be administered to generate asystemic response first, followed by a mucosal response. Becauseprolonged antigen stimulation can be achieved with DNA vaccination, itis possible that a single dose, or a limited number of doses of thevaccine will be adequate to achieve protection. DNA is a relativelystable molecule, facilitating storage and handling. Finally, the ease ofmanufacture, scale-up and distribution of DNA vaccines makes thistechnology very attractive. One example of an HIV DNA vaccine is thepVacc10 HIV DNA vaccine. The generation of the pVacc10 HIV construct isdescribed in Example 1.

In one embodiment of the instant invention, a nucleic acid constructencodes mutant HIV particles that produce non-infectious HIV particleswhen expressed in mammalian cells or injected into primates. Theconstruct has a number of mutations in the NC and RT and/or In genes ofa wild-type HIV genome. Specifically, the nucleocapsid gene is mutated,such that the amino acid sequence of the encoded protein is altered,wherein the alteration occurs in at least one amino acid selected fromthe group consisting of lysine 14, lysine 20, arginine 26, arginine 29,arginine 32, lysine 33, lysine 34, lysine 38, lysine 41, lysine 47 andcombinations thereof. Moreover, either the reverse transcriptase gene,or the integrase gene, or both is mutated, such that the amino acidsequence of the encoded protein is altered. Mutations of the reversetranscriptase gene that result in an alteration of the amino acidsequence of the encoded protein occur in at least one amino acidposition selected from the group consisting of tryptophan 71, arginine72, arginine 78 and combinations thereof. Mutations in the integrasegene that alter the amino acid sequence of the encoded protein occur inat least one amino acid selected from the group consisting of histidine14, histidine 18, cysteine 42, cysteine 45, aspartic acid 66, asparticacid 118, glutamic acid 154 and combinations thereof.

The nucleotide sequence of mutated RT is shown SEQ ID NO: 27 and thecorresponding amino acid sequence of the mutated RT is shown in SEQ IDNO: 28 displaying the mutation at positions 71, 72 and 78. Thenucleotide sequence of mutated In is shown in SEQ ID NO: 29 and thecorresponding amino acid sequence of the mutated In is shown in SEQ IDNO: 30 displaying the mutation at positions 14, 18,42, 45, 66, 118 and154. The nucleotide sequence of the mutated NC is shown in SEQ ID NO: 31and the corresponding amino acid sequence of the mutated NC is shown inSEQ ID NO: 32 displaying the mutation at positions 14, 20,26, 29, 32,33, 34, 38, 41, and 47. The nucleotide sequence of pVacc10 constructcomprising all the mutations, as shown in SEQ ID NO: 33.

III. Recombinant Technologies

The instant invention relates to nucleic acid molecules encoding mutatedHIV molecules. This invention relates to DNA constructs comprising anucleic acid molecule encoding HIV proteins operatively linked torecombinant host cells. As appropriate, nucleic acid molecules of thepresent invention can be RNA, for example, mRNA, or DNA, such as cDNAand genomic DNA. DNA molecules can be double-stranded orsingle-stranded; single stranded RNA or DNA can be either the coding, orsense, strand or the non-coding, or antisense, strand. The nucleotidesequence can include at least a fragment of the amino acid codingsequence along with additional noncoding sequences such as introns andnon-coding 3′ and 5′ sequences (including regulatory sequences, forexample). Additionally, the nucleotide sequence can be fused to a markersequence, for example, a sequence which encodes a polypeptide to assistin isolation or purification of a viral protein(s). Such sequencesinclude, but are not limited to, those which encode aglutathione-S-transferase (GST) fusion protein and those which encode ahemagglutin-A (HA) peptide marker for influenza. Various promoters havebeen engineered to drive expression of genes, such as the HIV genes, asdifferent promoters might produce different levels of antigen in thevarious cells targeted by DNA vaccination. All constructs of the presentinvention contain multiple CpG motifs, which are associated withimproved immunostimulatory properties of DNA vaccines. (Krieg, TrendsMicrobiol. 7:64–65 (1999), the teachings of which are incorporatedherein in their entirety by reference).

The invention provides expression vectors containing a nucleic acidsequence encoding one or more viral proteins described herein. Many suchvectors are commercially available, and other suitable vectors can bereadily prepared by the skilled artisan. The invention also relates toexpression vectors comprising the nucleic acid molecule encoding thevirus which are transfected into host cells, such as bacterial cells,insect cells, yeast cells, avian cells, fungal cells, plant cells,insect cells and mammalian cells. It should be understood that thedesign of the expression vector may depend on such factors as the choiceof the host cell to be transformed and/or the type of protein desired tobe expressed. For instance, the proteins of the present invention can beproduced by ligating the HIV nucleic acid sequence, or a portionthereof, into a suitable vector for expression in either prokaryoticcells, eukaryotic cells, or both. (See, for example, Broach, et al.,Experimental Manipulation of Gene Expression, ed. M. Inouye (AcademicPress, 1983) p. 83; Molecular Cloning: A Laboratory Manual, 2^(nd) Ed.,ed. Sambrook et al. (Cold Spring Harbor Laboratory Press, 1989) Chapters16 and 17, the teachings of which are herein incorporated in theirentirety by reference). Typically, expression vectors will contain oneor more selectable markers, including, but not limited to, the gene thatencodes dihydrofolate reductase and the genes that confer resistance toneomycin, tetracycline, ampicillin, chloramphenicol, kanamycin andstreptomycin resistance.

Eukaryotic or prokaryotic host cells transfected by the describedvectors are also provided by this invention. For instance, cells whichcan be transfected with the vectors of the present invention include,but are not limited to, mammalian cells, such as Chinese hamster ovarycells (CHO), COS cells, CEM leukemic lymphocytes, MCF-7 breast cancercells, H9 and 293T cells.

Thus, a nucleotide sequence described herein can be used to produce arecombinant form of a viral protein via an eukaryotic cellularprocesses. Similar procedures, or modifications thereof, can be employedto prepare recombinant peptides according to the present invention bymicrobial means or tissue/cell culture technology. Accordingly, theinvention pertains to the production of described peptides byrecombinant technology.

The present invention pertains to the transfection of host cells with amutated viral genome. There are several methods of transfection that oneof ordinary skill in the art may employ. For example, transfection canbe accomplished by calcium phosphate precipitation. In this method, aprecipitate containing calcium phosphate and DNA is formed by slowlymixing a HEPES-buffered saline solution with a solution containing about2.5 M calcium chloride and about 10 to 50 μg of DNA. This precipitateadheres to the surface of the host cell. The additional use of about 10%solution (vol/vol) of glycerol or dimethyl sulfoxide will increase theamount of DNA absorbed in some host cells. (Ausubel et al. (eds.),Current Protocols in Molecular Biology, Greene Publishing Associates andWiley-Interscience, 5^(th) ed., 1991), vol. 1, pp. 9.1.1–9.1.3, theteachings of which are herein incorporated in their entirety byreference).

Transfection may also be accomplished by electroporation of the hostcell. Electroporation can be used for both transient and stabletransfection. The host cell is placed in suspension and put into anelectroporation cuvette and then the DNA is added. The cuvette isconnected to a power supply, and the cells are subjected to ahigh-voltage pulse of defined magnitude and length, for example, shocksat 1 to 2 kV with a 3-μF capacitance is employed. The cell is thenallowed to recover briefly before the media is changed. (Ausubel, et al.(eds.), Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley-Interscience, 5^(th) ed., 1991), vol. 1, pp.9.3.1–9.3.4, the teachings of which are herein incorporated in theirentirety by reference). Both of these transfection methods, calciumprecipitation and electroporation, are known to one skilled in the art.

Transfection may also be liposome-mediated. This method is well known tothe skilled artisan. Generally, using liposomes to deliver DNA intodifferent cell types results in higher efficiency and greaterreproducibility than other transfection methods. Essentially, plasmidDNA derived from either crude (miniprep) or purified (through cesiumchloride centrifugation) preparations is mixed with a commerciallyavailable liposome suspension comprising cationic lipids. TheDNA-liposome complex is applied to approximately 5×10⁵ cells/well andgrown overnight in a CO₂ incubator at about 37° C. to around 80%confluency. after overnight incubation with the liposome-DNA complex,the cells can be harvested by scrapping, trypsinization, or freeze thawmethods as described by Ausubel et al. (eds.), Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley-Interscience,5^(th) ed., 1991), vol. 1, pp. 9.4.1–9.4, the teachings of which areherein incorporated in their entirety by reference.

IV. Codon Optimization and Homology

Also included within the scope of the invention are conservativemutations. For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. conservativemutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cystine, serine, threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids. In similar fashion, the amino acid repertoirecan be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine,isoleucine, serine, threonine, with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine,tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6)sulfur-containing=cysteine and methoinine. (See e.g., Biochemistry, 2nded, Ed. by L. Stryer, W H Freeman and Co.: 1981).

DNA molecules that code for the viral peptides can easily be determinedfrom the list of codons in Table 3, below. In fact, since there is afixed relationship between DNA codons and amino acids in a peptide, anydiscussion in this application of a replacement or other change in apeptide is equally applicable to the corresponding DNA sequence or tothe DNA molecule, recombinant vector, transformed microorganism, ortransfected eukaryotic cells in which the sequence is located (and viceversa). An important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed. Therefore, anumber of different nucleotide sequences may code for a given amino acidsequence. Such nucleotide sequences are considered functionallyequivalent since they can result in the production of the same aminoacid sequence in all organisms, although certain strains may translatesome sequences more efficiently than they do others.

TABLE 3 The genetic code GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG,GCT Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N)AAC, AAT Aspartic acid (Asp,D) GAC, GAT Cysteine (Cys, C) TGC, TGTGlutamic acid (Glu,E) GAA, GAG Glutamine (Gln, Q) CAA, CAG Glycine (Gly,G) GGA, GGC, GGG, GGT Histidine (His, H) CAC, CAT Isoleucine (Ile, I)ATA, ATC, ATT Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys,K) AAA, AAG Methionine (Met, M) ATG Phenylalanine (Phe,F) TTC, TTTProline (Pro, P) CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC,TCG, TCT Threonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGGTyrosine (Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTTTermination signal TAA, TAG, TGA (end) Key: Each 3-letter tripletrepresents a trinucleotide of DNA having a 5′ end on the left and a3′ end on the right. The letters stand for the purine or pyrimidinebases forming the nucleotide sequence. A = adenine, G = guanine, C= cytosine, T = thymine

Codons can be chosen for use in a particular host organism in accordancewith the frequency with which a particular codon is utilized by thathost, if desired, to increase the rate at which expression of thepeptide occurs, a process known as “codon optimization.” Codon bias hasbeen observed in many species. The preferential use of codons for agiven gene is positively correlated with its expression efficiency.Codons are DNA triplets that constitute “one information unit” forprotein synthesis; 64 distinct triplet sequences exist. The 20 aminoacids are encoded by 61 codons and the remaining 3 codons encode proteinsynthesis termination signals. Each codon is specific for a single aminoacid. In most cases, however, a single amino acid, can be identified bymore than one codon. Thus different DNA sequences can code for the sameprotein. Not all codons are used with the same frequency betweenspecies. Some codons are more commonly used while others are not. Codonswhich are “rare” usually correlate with a reduced intracellular level ofaminoacyl-tRNAs, the biochemical building blocks used by ribosomes tomatch a codon with the correct amino-acid during protein synthesis. If aparticular aminoacyl-tRNA is deficient, then use of the correspondingcodon will slow the rate of protein synthesis and can decrease yield ofa desired protein product. (See e.g., Sharp et al., Nucleic Acids Res.,17:5029–39 (1989), the teachings of which are herein incorporated intheir entirety by reference).

The relative frequency of use for each codon can vary significantlybetween species. Codon usage for a wide variety of organisms iscataloged by the National Institute of Health of Japan. Engineering aconstruct in which the codons have been optimized would result in adifferent DNA sequence that has been uniquely optimized for expressionof the desired protein sequence in any host cell. Accordingly, nucleicacid constructs that have been codon optimized are intended to be withinthe scope of the invention.

The terms “codon optimization” and/or “codon optimized for humans” areused herein to encompass various synthetic gene sequences which encodeproteins, such as the NC, RT and IN proteins of the non-infectious viralparticles of the present invention, wherein at least one non-preferredor less preferred codon in the natural gene encoding the protein hasbeen replaced by a preferred codon encoding the same amino acid.

For increased human expression the preferred codons can include one ormore of the following: Ala (gcc); Arg (cgc); Asn (aac); Asp (gac) Cys(tgc); Gln (cag); Gly (ggc); His (cac); Ile (atc); Leu (ctg); Lys (aag);Pro (ccc); Phe (ttc); Ser (agc); Thr (acc); Tyr (tac); and Val (gtg).Less preferred codons will typically include one or more of thefollowing: Gly (ggg); Ile (att); Leu (ctc); Ser (tcc); Val (gtc). Ingeneral, the degree of preference of particular codon is indicated bythe prevalence of the codon in highly expressed human genes asindicated, for example, in U.S. Pat. No. 5,795,737, herein incorporatedby reference. For example, “atc” represents 77% of the Ile codons inhighly expressed mammalian genes and is the preferred Ile codon; “att”represents 18% of the Ile codons in highly expressed mammalian genes andis the less preferred Ile codon. The sequence “ata” represents only 5%of the Ile codons in highly expressed human genes and thereof is anon-preferred codon.

Replacing a codon with another codon that is more prevalent in highlyexpressed human genes will generally increase expression of the gene inmammalian cells. Accordingly, the invention includes replacing a lesspreferred codon with a preferred codon as well as replacing anon-preferred codon with a preferred or less preferred codon. A“synthetic gene” is a nucleotide sequence encoding a naturally occurringprotein in which a portion of the naturally occurring codons have beenreplaced by other codons. For example, a non-preferred codon is replacedwith preferred codon or a less preferred codon encoding the same aminoacid. In addition a less preferred codon can be replaced by a preferredcodon. Synthetic genes generally encode proteins normally expressed byeukaryotic cells, including mammalian cells. However, by replacingcodons to create a synthetic gene the expression in mammalian cells(especially human cells) of a viral genes described herein can beincreased compared to the expression of the naturally occurring gene.

In preferred embodiments, the synthetic gene is capable of expressingthe NC, RT or IN gene protein at a level which is at least 110%, 150%,200%, 500%, or more of that expressed by said natural gene underidentical conditions (i.e., same cell type, same culture conditions,same expression vector). In the present invention the codon bias presentin the HIV gag and pol proteins can be overcome by replacement of aportion of the non-preferred and/or less preferred codons found in theHIV genes with preferred codons to produce a vector capable of higherlevel expression.

Accordingly, in one embodiment, preferred codons for the viralconstructs of the present invention can be selected from the groupconsisting of gcc, cgc, aac, gac, tgc, cag, ggc, cac, atc, ctg, aag,ccc, ttc, agc, acc, tac, and gtg, while less preferred codons can beselected from the group consisting of ggg, att, ctc, tcc, and gtc andall codons other than the preferred codons and the less preferred codonscan be considered non-preferred.

Codon optimization of the HIV-1 gag-pol gene has also been described byKotsopoulou et al. who has constructed a HIV-1 gag-pol gene where thenucleotide sequence has been altered in the majority of the codons toretain the primary amino acid sequence, but to exploit the favored codonusage of human cells. (Kotsopoulou et al. J. Virol. (2000) 74:4839–4852.) Codon optimization of the gag gene has been described byGraf et al. who found increased expression of the gag protein inmammalian cells (Graf et al. J. Virol. (2000) 74: 10822–10826. Inaddition, codon optimization has been demonstrated to increase theexpression levels and the immunogenicity of DNA vaccines encoding gag,as described by Deml et al. J. Virol. (2001) 75: 10991–101001.

It will be appreciated by those skilled in the art that one or more DNApolymorphisms that lead to changes in the amino acid sequence are withinthe scope of the invention. Such genetic polymorphisms may exist due tonatural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms that are a result of natural allelicvariations and that do not alter the functional activity of thedifferent HIV regions, are also intended to be within the scope of theinvention.

EXAMPLES Example 1 Generation of the pVacc10 Construct

The parental DNA clone used in the development of pVacc10 is the plasmidpM1–2/BR which was a derivative of the biologically active pHXB2gpt.(See Poon et al., J. Virol. 70 (10): 6607–6616 (1996), the teachings ofwhich are incorporated herein in their entirety by reference). In orderto construct a plasmid with HIV-1 sequences, SIV sequences in pVacc3were substituted with HIV-1 sequences using the Nar 1-Xho1 fragment(where the Xho11 site has been filled in by Clainow polymerase) frompM1–2/BR which replaced a Nar1-SnaB1 fragment in pVacc3. Thismanipulation resulted in a plasmid carrying HIV-1 coding sequences underthe control of the CMV promoter instead of the HIV-1 LTR. Furthermore,the 3′ HIV-1 LTR was no longer present and it was substituted by theSV40 polyA. In this vector, arginine and lysine of the HIV-1 NC(nucleocapsid) gene were mutated to alanines. Oligo-mediatedsite-specific mutagenesis by overlap extension was performed on pHXB2gptto obtain pM1–2/BR. (See Horteon et al., “Methods in Enzymology,”217:270–279 (1993), Academic Press, San Diego, the teachings of whichare incorporated herein in their entirety by reference). For each codonmutated to encode an alanine, the first two bases were changed to a G(guanine) and a C (cytosine) while the third base was left unaltered.Additionally, a total of 12 mutations were introduced in the RT andIntegrase genes using the QuickChange Mutagenesis kit (Statagene).Oligonucleotides used for the mutagenesis correspond to the forward andreverse (complementary, (C)) primers for the different HIV genes. Theunderlined regions indicate the points of mutation.

  (a) RT mutagenesis: Forward: HIV-2750 5′cagtactaaagcggcaaaattagt (SEQ.ID. NO:11) agatttcgcagaacttaat Reverse:HIV-2750C5′attaagttctgcgaaatctacta (SEQ. ID. NO:12) attttgccgctttagtactg  (b) Integrase mutagenesis: Forward: HIV-4250 5′ggcccaagatgaagctgagaaat(SEQ. ID. NO:13) atgccagtaattgg Reverse:HIV-4250C5′ccaattactggcatatttctcag (SEQ. ID. NO:14) cttcatcttgggccForward: HIV-4338 5′gtagccagcgct-gataaagct (SEQ. ID. NO:15) cagctaaaaggReverse: HIV-4338C5′ccttttagctgagctttatcagc (SEQ. ID. NO:16) gctggctacForward: HIV-4411 5′ggcaactagcttgtacacattta (SEQ. ID. NO:17) gaaggReverse: HIV-4411C5′ccttctaaatgtgtacaagctag (SEQ. ID. NO:18) ttgccForward: HIV-4564 5′caatacatactgccaatggcagc (SEQ. ID. NO:19) Reverse:HIV-4564C5′gctgccattggcagtatgtattg (SEQ. ID. NO:20) Forward: HIV-46745′ggagtagt-agcatctatgaata (SEQ. ID. NO:21) aag Reverse:HIV-4674C5′ctttattcatagatgctactact (SEQ. ID. NO:22) cc (c) Nucleocapsidmutagenesis: Forward pBR 5′-AATTGCGCGGCCCCTGCGGCAGCGGGC (SEQ. ID. NO:23)TGT Reverse: pBRC 5′-ACAGCCCGCTGCCGCAGGGGCCGCGCA (SEQ. ID. NO:24) ATTForward: pM1-2 5′-AGGGCCCCTAGGAAAAAGGGCTGTT (SEQ. ID. NO:25)GGGCATGTGGAGCGGAAGGACACCAAATGGCAG ATTGTACTGAG Reverse: pM1-2C5′-GCCCTTTTTCCTAGGGGCCCTGCA (SEQ. ID. NO:26)ATTTGCGGCTGTGTGCCCTTCTGCGCCACAATTG AAACACGCAACAATCTT

All mutations were confirmed by dideoxy sequencing. All DNAmanipulations were performed according to standard procedures. (Ausubelet al., (1987) Current Protocol in Molecular Biology, John Wiley andSons, Inc., NY, the teachings of which are incorporated herein in theirentirety by reference).

Example 2 Production of Viral Particles Using pVAcc10

To investigate the production of viral particles generated using thepVacc10 construct, recombinant cell lines were created. One example of arecombinant cell line was created using the cell line 293T. The cellline 293T was maintained in Dulbecco modified Eagle medium (GIBCO/BRL,Bethesda, Md.) supplemented with 10% fetal bovine serum (GIBCO) at 37°C. under 5% CO₂. Another cell-line, the H9 T-lymphoid cell line was alsoused and maintained as previously described. (Hanke, T., et al., 1999.J. Virol. 73(9): 7524–7532, the teachings of which are incorporatedherein in their entirety by reference). Transfection of 293T cells bycalcium phosphate precipitation and analysis of viral mutants werecarried out as described previously. (Aldovini et al., J. Virol. 64:1920–1926 (1990), and Almond et al., AIDS 12 (Suppl A): S133–140 (1998),the teachings of which are incorporated herein in their entirety byreference). Supernatants from transfected 293T cell cultures wereharvested 48 hours post-transfection and assayed for p24 antigen usingan enzyme-linked immunosorbent assay (ELISA) (p24 core profile kit,DuPont) and for RT activity (as described by Bernstein et al., Vaccine17:1681–1689 (1999), the teachings of which are incorporated herein intheir entirety by reference). Viral supernatants derived from twoindependent transfections of pVacc10 were tested in infectivity assays.Cells, specifically 5×10⁵ H9 cells, were exposed to amounts of virusfrom transfected cells equivalent to 25 ng of p24 in 2 ml of medium.After 3 hours of infection, cells were washed, resuspended in 2 ml oftissue culture medium and maintained in 24 well plates. Cultures werefed every 4 days by removing 1.5 ml of the 2 ml of cell culturesuspension and replacing it with fresh medium. Cell density increasedfrom 5×10⁵ just after feeding to 2×10⁶ four days later. At each 4-dayinterval, cleared supernatants were utilized for p24 ELISA and harvestedcells were assayed by immunofluorescence. Each time point of eachculture was evaluated in duplicate in both assays. Cultures were carriedfor 30 days after infection. Viral particles produced by pVacc10 werenot infectious in culture.

Example 3 Investigation of RNA Viral Packaging Using pVacc10

To investigate whether RNA was packaged using the pVacc10 construct thefollowing procedure was used. After evaluation of p24 in the medium,supernatant from cells transfected with pVacc10 containing virusparticles equivalent to 15 ng of p24 was centrifuged to pellet thevirions, viral RNA was extracted and quantitative RT-PCR was performedon RNA samples according to a previously described procedure. (Fuller etal., Immunol. Cell Biol. 75(4):389–396 (1997), the teachings of whichare incorporated herein in their entirety by reference). Briefly, thepellet was resuspended in 0.5 ml of Solution D (4.2 M guanidinethiocyanate, 0.1 M sodium citrate, 0.5% SDS, 7.2% 2-mercaptoethanol)containing 120 mg/ml of yeast tRNA as a carrier to monitor final RNArecovery. The RNA was extracted with phenol-chloroform and precipitatedwith ethanol. In order to eliminate contaminating transfection orcellular DNA, the RNA was then treated with 16 units of RQ1 DNase I(Promega) in the buffer recommended by the manufacturer (40 mM Tris,pH8, 10 mM NaCl, 6 mM MgCl₂, 10 mM CaCl₂) in the presence of 80 units ofrecombinant RNasin Ribonuclease Inhibitor (Promega) for 1 hour at 37° C.The DNase I was denatured with Solution D and the RNA was precipitatedwith ethanol. DNase I-treated viral RNAs were resuspended in diethylpyrocarbonate (DEPC)-treated water and the yeast tRNA concentration wasadjusted to 0.5 mg/ml. RNA samples were obtained from three independenttransfections. RNA samples corresponding to 2 ng of p24 were reversetranscribed in a 30 μl reaction with Superscript II™ (GIBCO/BRL) using agag-specific primer (HIVc1686, 5′-ACCGGTCTACATAGTCTCTA-3′; SEQ. ID. NO.34). Three ml of this reaction were subjected to PCR with AmpliTaq™ DNAPolymerase (Perkin Elmer), in the presence of ³²P-dCTP, employing thesame primer used in the RT reaction and paired with an upstreamgag-specific primer (HIV979, 5′-TACAACCATCCCTTCAG-3′; SEQ. ID. NO: 35).The negative controls included a sample from an RT-PCR reaction lackinginput RNA, and an RT-PCR reaction with RNA extracted from a mocktransfected supernatant. A PCR reaction on an equivalent amount of RNAwhich did not undergo reverse transcription was carried out for eachsample to exclude incomplete DNase I treatment. An RT-PCR reaction usingactin-related primers (ACT1, 5′-ATGGAAGAAGAGATCCGC-3′ (SEQ. ID. NO: 36)and ACTR2, 5′-CCTCGTAGATGGGCACCG-3′ (SEQ. ID. NO: 37)) was carried outto eliminate cellular RNA contamination. Equal volumes of RT-PCR or PCRsamples were subjected to polyacrylamide gel electrophoresis (PAGE) andautoradiography. The intensity of each band was quantitated using aMolecular Dynamics PhosphorImager with ImageQuant software (MolecularDynamics). The viral particles generated by pVacc10 do not incorporatedetectable viral RNA.

Example 4 Vector Construction for SIV-Based Systems

To further determine the in-vivo efficacy of the vaccination protocoland the generation of the immune response, the art-recognized SIV-basedexperimental system was employed. The SIV constructs have the samemutations in the same amino acid residue positions as the pVAcc10construct. In particular, mutations were made in the nucleocapsidprotein at amino acid positions lysine 14, lysine 20, arginine 26,arginine 29, arginine 32, lysine 33, lysine 34, lysine 38, lysine 41,and lysine 47; in the RT protein at amino acid positions tryptophan 71,arginine 72, and arginine 78; in the In protein at positions histidine14, histidine 18, cysteine 42, cysteine 45, aspartic acid 66, asparticacid 118, and glutamic acid 154.

The same rationale was employed using the SIV virus as was employed forthe HIV virus. Specifically, certain mutations were made in the SIVgenome in order to produce an SIV virus that was incapable of infectionbut that could stimulate a host's immune system, in this case the hostwas the rhesus macaques monkey. To summarize the results presentedbelow, the protocol established herein for making a viral DNA-basedvaccine resulted in the stimulation of the specific-immune system,including the elaboration of specific IgA molecules particular to themucosa.

All mutants of SIVmac239 were constructed using the infectious clonepMA239 (14110 bp) which carries a full copy of the molecular clone ofSIV mac239. Mutations were introduced into the nucleocapsid (NC),reverse transcriptase (RT) and integrase (IN) genes of the SIV genomeusing oligonucleotide-mediated site directed mutagenesis by overlappingextension PCR (Table 4). (See Horton, R. M., et al., In: R. Wu (ed.).Methods in Enzymology Vol. 217 Part H (1993), Academic Press, San Diego,Calif., the teachings of which are incorporated herein in their entiretyby reference).

TABLE 4 SIV proviral DNA constructs construct 5′end Gag NC RT IN 3′endpMA239 LTR wt wt wt nef-stop, 3′ LTR pMA22polyA LTR 12 mutations¹ 3mutations² 7 mutations³ Δnef, polyA⁴ pVacc 1 CMV⁵ 12 mutations 3mutations 7 mutations Δnef, polyA pVacc 2 EF1a⁶ 12 mutations 3 mutations7 mutations Δnef, polyA ¹The NC mutations are Q3:G, C3:W, five R:A, andfive K:A. ²The RT mutations are W71:A, R72:A, and R78:A. ³The INmutations are alanine substitutions of the histidine and cysteine in theHHCC domain and of the aspartic and glutamic acid residues in the DD35Edomain. ⁴The SV40 polyadenylation site replaces the 3′end of the SIVgenome from the termination codon of the env gene to the end of the 3′LTR. ⁵The cytomegalovirus promoter (CMV) replaces the SIV 5′ LTR fromthe beginning of the SIV sequence in pMA239 to the SIV Narl site inpVacc1. ⁶The eukaryotic polypeptide chain elongation factor 1a promoter(EF1a) replaces the SIV 5′ LTR from the beginning of the SIV sequence inpMA239 to the SIV Narl site in pVacc2.The oligonucleotides used in the mutagenesis are listed in Table 5(oligonucleotides #1–18).

TABLE 5 Oligonucleotide primers used in generation of recombinantviruses (Codon mutations or restriction endonuclease sites introduced inthe primer for construction of SIV recombinant are underlined). PrimerSequence  1. SIV2103 5′-ATGGATCCAACTGGGGTTGCA  2. SIV2517C/NC5′-TGCAGAGTGTCCCTCTGCCCCACAAT TCCAACACGCAATTGGCGCTGCTGGTCCCGC CTGTTC  3.SIV2506/NC 5′-GAGGGACACTCTGCAGCGCAATGCGCAGCCCCAGCAGCACAGGGATGCTGGGCATGGGG AGCAATGGAC  4. SIV2709C5′-CACAGCTGGGTCCTCTGGGGGAG  5. SIV2630 5′-GAAAGAAGCCCCGCAATTTCC  6.SIV3347C/RT 5′-TAGTTCCGCAAAATCTATCAGCATTGCC GCTTTGTTGTTATC  7.SIV3309/RT 5′-AAGAACAAAGCGGCAATGCTGATAGA TTTTGCGGAACTAAAT  8. SIV3632C5′-ATTTGCCTTCCTGAAGGGTTC  9. SIV4513 5′-AACAGACTACTAATCAACAAG 10.SIV4880C/INT-HH 5′-CACTATTCTGGGTAATCCAAATTTGAATACCAATTCTTTTACATTACTAGCGTATTTAT CAGCTTCTTCTTGT 11. SIV4857/INT-CC5-AAATTTGGATTACCCAGAATAGTGGCCAG ACAGATAGTAGACACCGCTGATAAAGCTCAT CAGAA12. SIV5464C 5′-TAATAGACCCGAAAATTTTTA 13. SIV4964/INT-D5′-TTGGCAAATGGCTTGTACCCATCT 14. SIV4986C/INT-D5′-GATGGGTACAAGCCATTTGCCAAG 15. SIV5120/INT-D5′-TCTACACACAGCTAATGGTGCTAA 16. SIV5142C/INT-D5′-TAGCACCATTAGCTGTGTGTAGAT 17. SIV5228/INT-E5′-GGGAGTAGTGGCAGCAATGAATCA 18. SIV5250C/INT-E5′-GATTCATTGCTGCCACTACTCCCT 19. A22/Blpl 5′-AACAGCTTAGCTCTAGAGTCGACCAGACAT 20. A22C/Snabl 5′-AACATACGTATATTAAAGCAGTACTTGT TA 21. CMV4062/Sfl5′-TTGCTCACATGGCCTCAGAGGCCTTCAA TATTGGCC 22. CMV824C/BspEl5′-TCGAGACTGTTGTGTCCGGAGCACTGAC TG 23. EF522/Sfl5′-AATGGACCTTCTAGGGCCTCAGAGGCCT GG 24. EF1770C/BspEl5′-TCTCGAGGTCGAGGGATCTCCGGAGAAT TCTTCACGACA

In addition, the SIV 5′ LTR was replaced by the Cytomegalovirus promoter(CMV) or eukaryotic polypeptide chain elongation factor 1a promoter(EF1a) and the SIV 3′ LTR was substituted with the polyadenylation site(polyA) from pSG5 (Stratagene). Paired primers, 19 and 20, (Table 5)were used to obtain the polyA fragment from the pSG5 vector by usingPCR, which was used as a template. The fragment was digested withBlpI/SnaBI and ligated to a SnaBI site that was introduced in thewild-type proviral DNA to replace sequences 9505–10709 of SIVmac239 andto the unique BlpI site present in pMA239. To change the SIV 5′ LTR to aCMV promoter, primers 23 and 25 (adding, respectively, SfiI and BspEIsites at the two ends of the fragment) were used to obtain the CMVpromoter fragment from the pRL CMV vector. (See Regier et al., AIDS Res.Hum. Retroviruses 6:1221–1231 (1990), the teachings of which areincorporated herein in their entirety by reference). A SfiI site wasintroduced in the pUC18 sequence of pMA239, and this site was introduced220 nucleotides 3′ of the AatII site. The fragment containing the CMVpromoter replaced the sequences from the SfiI site to the NarI site ofpMA239. The modified plasmid is designated Vacc1 and contains the CMVpromoter, multiple mutations in the SIV structural genes, and areplacement of the 3′ LTR with a polyA site.

A similar strategy was used to replace the 5′ LTR with an EF1a promoter(NCBI Accession No. AF16376). Primers 26 and 27 were used to amplify theEF1a promoter from pEBB by PCR. (See Tanaka et al., Mol. Cell. Biol.15:6829–6837 (1995), the teachings of which are incorporated herein intheir entirety by reference). The fragment obtained replaces thefragment from the SfiI site to the NarnI site of pVacc1, to obtain theplasmid, designated pVacc2. All mutated viral sequences were confirmedby dideoxy sequencing, and the DNA manipulations were carried outaccording to previously published procedures. (Ausubel et al., CurrentProtocol in Molecular Biology, John Wiley and Sons, Inc., New York(1987), the teachings of which are incorporated herein in their entiretyby reference).

Example 5 Transfection of Cells Using the SIV Construct

Eukaryotic cell transfections: 293T cells were seeded at a density of10⁶/100-mm plate, 24 hours prior to transfection, in DME mediumsupplemented with 10% fetal calf serum and incubated at 37° C. in 5%CO₂. Transfection was generally carried out by a calcium phosphatemethod using 10 μg of plasmid DNA/100 mm plate as described by Chen andOkayama. (Chen et al., Mol. Cell. Biol. 7:2745–2752 (1987), theteachings of which are incorporated herein in their entirety byreference). Transfection efficiency was evaluated after 48 hours byimmunoperoxidase staining of transfected cells using an SIV-specificpolyclonal serum and a rabbit-anti monkey IgG conjugated with peroxidase(Sigma).

Example 6 Nucleic Acid Analysis

RNA was extracted from 293T cells using the Triazol reagent (Gibco/BRL).In order to eliminate contaminating transfection or cellular DNA, theRNA was treated with 16 units of RQ1 DNase I (Promega) in the bufferrecommended by the manufacturer and quantitative RT-PCR was performed onRNA samples according to a previously described procedure. (Poon et al.,J. Virol. 72:1983–1993 (1998), the teachings of which are incorporatedherein in their entirety by reference). Signals were detected byautoradiography with Kodak XAR film using a Dupont Quanta III screen.The intensity of each band was quantitated using a Molecular DynamicsPhosphorImager with ImageQuant software (Molecular Dynamics).

Example 7 Particle Characterization

Particles produced from the vectors were characterized biochemically.Supernatants from transfected cells were collected, filtered through a0.45 micron filter and centrifuged through a 3 ml cushion of 15%(w/vol.) sucrose at 27,000 rpm for 3 hours in an SW28 rotor (Beckman).Each pellet was resuspended in 10 mM Tris, (pH6.8) with 0.1% Triton andthe p27 Capsid Antigen (CA) content determined by ELISA assay. Samplesequivalent to approximately 25 ng of CA were resuspended in Laemmlibuffer (5% glycerol, 1% SDS, 31.875 mM Tris (pH6.8), 0.005% bromophenolblue), subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). The proteins were transferred tonitrocellulose, and probed with SIV positive serum as describedpreviously. (Poon et al., J. Virol. 72:1983–1993 (1998), the teachingsof which are incorporated herein in their entirety by reference).¹²⁵I-Protein A (New England Nuclear) was used to detect HIV specificproteins by autoradiography.

After evaluation of SIV CA in the medium of all the transfectants,supernatants containing virus particles equivalent to 250 ng of CA werecentrifuged to pellet the virions. Viral RNA extraction was carried outin presence of equal amounts of added tRNA, which was used to monitorthe final recovery of RNA. RNA samples were resuspended in DEPC treatedH₂O to obtain identical concentrations of tRNA (1 μg/μl). QuantitativeRT-PCR was performed on RNA samples equivalent to 4 ng of p27 accordingto a previously described procedure. (Poon et al., J. Virol. 70:6607–6616 (1996), the teachings of which are incorporated herein intheir entirety by reference). Electron microscopy (EM) was carried outon sections of embedded particles according to standard procedures.

Example 8 Infectivity Assays

Viral supernatants derived from two independent transfections perconstruct were tested in infectivity assays. Cells, specifically 5×10⁵CEMx174 cells, were exposed to amounts of virus from transfected cellsequivalent to 250 ng of p27 in 2 ml of medium. After 3 hours ofinfection, cells were washed, resuspended in 2 ml of tissue culturemedium and maintained in 24-well plates. Cultures were fed every 4 daysby removing 1.5 ml of the 2 ml cell culture suspension and replaced withfresh medium. Cell density increased from 5×10⁵ just after feeding toapproximately 2×10⁶ 4 days later. At each 4 day interval, clearedsupernatants were tested for virus content by SIV p27 ELISA. Cultureswere maintained for 30 days after infection. Nested PCR was performed oncellular DNA, and RT-PCR on RNA, from pelleted supernatants fromcultures that scored negative in SIV p27 ELISA.

Example 9 Vaccine Formulation Using the SIV Constructs

The candidate vaccine plasmids were grown from a single E. coli colonyin Luria Broth with 100 μg/ml ampicilin at 30° C. for no more than 18hours. DNA was purified by CsCl gradient followed by passage through anendotoxin-free column (Quiagene). (See Ausubel et al., Current Protocolin Molecular Biology, John Wiley and Sons, Inc., New York (1987), theteachings of which are incorporated herein in their entirety byreference). The vaccines were formulated in a variety of different ways.For intradermal and intramuscular administration of the DNA vaccine,saline solution (Sigma) was used to resuspend the DNA and theconcentration was adjusted to 1 μg/μl.

For mucosal administration, the vaccine DNA was formulated in liposomes,at a concentration of 0.5 mg/ml. Briefly, lyophilized DOTAP:Cholesterolin equimolar ratio (Calbiochem) was hydrated in 5% dextrose/H₂O (D5W) togive 20 mM DOTAP/20 mM Cholesterol (1×). (See Templeton et al., Nat.Biotechnol. 15:647–652 (1997), the teachings of which are incorporatedherein in their entirety by reference). The hydrated lipid mixture wassonicated for 5 minutes at 50° C. and heated for 10 minutes at 50° C.The mixture was sequentially extruded through decreasing size filtersfrom 1 to 0.1 μm. Whatman Anotop filters with an aluminum oxide membranespecially made for liposome extrusion were used. The liposome/DNAcomplexes were made by mixing together 60 μl of 20 mM liposomesuspension plus 90 μl of D5W, and 15 μl of a 10 mg/ml DNA stock plus 135μl of D5W.

For administration using a gene gun, the gold particle-associated DNApreparation was formulated according to the manufacturer protocol(Bio-Rad). Plasmid DNA was precipitated onto 1.6 μm gold particles at aDNA loading ratio (DLR) of 5 μg DNA/mg gold. The microcarrier loadingquantity (MLQ) was 0.5 mg gold/cartridge. The coated particles wereaccelerated using hand-held helium-powered Helios Gene Gun SyStem(BioRad).

Example 10 In Vivo Studies with Macaque Vaccination and Challenge

Nine Rhesus macaques were vaccinated at time 0, 9 weeks and 25 weekswith the SIV construct i.d. (group 1), i.d. and at the rectal mucosa(i.d/R) (group 2), i.d., R and i.m (group 3). When DNA was givenintradermally, the animals in all vaccine groups received 0.5 mg ofpVacc1 DNA (0.4 mg of DNA in saline by needle injection and 0.1 mg DNAby gene gun) in the skin that covers the gluteal area. For the mucosalvaccination of groups 2 and 3, 1 mg of pVacc1 DNA mixed with liposomes,prepared according to the protocol described above was administered tothe rectal mucosa approximately 5 cm from the anal verge with a smallsyringe without needle. The intramuscular vaccination of group 3consisted of the administration of 1 mg of vaccine pVacc2 DNA in 1 ml ofsaline in the gluteal muscle. When challenged, the macaques in thisexperiment received 5000 TCID₅₀ of cloned SIVmac239, administered to therectal mucosa by syringe without needle. This amount of virus isequivalent to 9 ng of p27 and is estimated to be approximately 10 rectalmacaque infectious doses (AID₅₀). This challenge dose corresponds to 10⁵AID₅₀ by i.v. titration.

Collection and Processing of Rectal Secretions

Rectal secretions were collected before and at intervals afterimmunization with absorbent Weck-Cel sponges (WQindsor BioMedical,Newton, N.H.) using a modified wicking method. (Kozlowski et al.,Comparison of the oral, rectal, and vaginal immunization routes forinduction of antibodies in rectal and genital tract secretions of women,Infect. Immun. 65:1387–1394 (1997), the teachings of which areincorporated herein in their entirety by reference). This tamponapplicator-based technique has been described in detail elsewhere.(Kozlowski et al., J. Acquir. Immune Defic. Syndr. 24(4):297–309 (2000),the teachings of which are incorporated herein in their entirety byreference). Briefly, a sponge moistened with 50 μl saline and housedwithin a pipet was inserted 6 cm into the rectum and exposed to rectalsurfaces by withdrawing the pipet 1.5 cm while holding the sponge inplace. After 5 minutes, the sponge was gently pulled back into thepipet, the entire apparatus was removed from the rectum, and spongeswere stored at −80° C. in Eppendorf tubes. To extract secretions, eachsponge was placed in the upper chamber of a double-chambered spinassembly soaked with 100 μl PBS containing 0.5% Igepal detergent (Sigma)and protease inhibitors then centrifuged at 20,000×g at 4° C. for 30minutes. The volume of secretion eluted from each sponge and dilutionfactors introduced by the pre-moistening saline and elution buffer werecalculated based on weights of fluid centrifuged into 2 mlmicrocentrifuge lower chamber tubes. Blood contamination in secretionswas assessed by using ChemStrips 4 (Boehringer-Mannheim) to measurehemoglobin, which in our hands was found negligible, representing only0.01% of that in blood on average.

Example 11 Antibody Detection

SIV-Specific IgG and IgA Detection

SIV-specific antibodies were measured by ELISA, using plates coated withwhole virus or purified virus-derived gp130. Incubations were performedin duplicate and multiple double dilutions of each sample wereevaluated. In assays for SIV-specific serum IgG antibodies, samples weretested initially at two dilutions (1:20 and 1:100). (Wyand et al., J.Virol. 70:3724–3733 (1996), the teachings of which are incorporatedherein in their entirety by reference). Samples highly positive at 1:100were re-tested and further diluted at 1:80, 1:160, 1:320, 1:640 and soon. Bound SIV-specific antibodies were detected by incubation with anaffinity purified donkey anti-human IgG-alkaline phosphatase conjugate(Jackson Laboratories).

For detection of SIV and gp130-specific IgA and IgG in rectal secretionsand virus-specific IgA in serum, Nunc MaxiSorp microtiter plates werecoated with 250 ng/well SIV viral lysate or purified native gp130 (bothfrom Advanced Biotechnologies, Rockville, Md.). Antibodies measured inthese SIV ELISAs likely do not include those to gp130 as this envelopeprotein could not be detected on plates coated with viral lysate using 5μg/ml of anti-gp130 antibody (Advanced Biotechnologies), the workingstandard in gp130 ELISAs. Plates were reacted at 4° C. with two-folddilutions of samples in 4% goat serum/0.05% Tween/PBS and developed thefollowing day with a highly specific anti-monkey IgA mouse IgGmonoclonal antibody (provided by Dr. Susan Jackson, University ofAlabama at Birmingham), followed by biotinylated goat anti-mouse IgGantibody (Southern Biotechnology Associates, Birmingham, Ala.) fromwhich antibodies cross-reactive with monkey IgG had been removed bypassage through a column of CNBr-activated Sepharose (Pharmacia)conjugated to monkey IgG. (See Ward et al., J. Med. Primatol. 24:74–80(1995), the teachings of which are incorporated herein in their entiretyby reference).

In assays for SIV-specific IgG in rectal secretions, plates weredeveloped with goat anti-monkey IgG antibody (Accurate, Westbury, N.Y.)that had been biotinylated in the laboratory using the Pierce (Rockford,Ill.) Sulfo-NHS-LC-Biotin EZ-link kit. Optimal dilutions of allantibodies were determined by checkerboard serial titration asdescribed. (Margulies, Criss-cross serial dilution analysis to determineoptimal reagent concentrations. In: Coico R., ed. Current Protocol inImmunology. New York: John Wiley & Sons, 1998:2.1.17–2.1.18, theteachings of which are incorporated herein in their entirety byreference). Color development was monitored after reacting plates withavidin-labeled peroxidase and ABTS/H₂O₂ substrate as described inKozlowski et al., (1997). Infect. Immun. 65:1387–1394. To determineendpoint titers of antibody in secretions, the last sample dilutionproducing an absorbance value≧mean absorbance+3 SD in 8 control wells(containing sample buffer with 4% goat serum in PBS-Tween) wasmultiplied by the dilution factor introduced into the secretion duringelution from sponges. Pooled serum from SIV-infected monkeys wasarbitrarily assigned 10,000 unit/ml of anti-SIV IgA antibody and used togenerate standard curves in these assays for interpolation of antibodyconcentrations in samples.

To determine with accuracy whether rectal secretions containedsignificant levels of SIV-specific antibodies and to facilitatecomparisons among animals in which total Ig concentations in secretionsare highly variable, measured antibody concentrations were divided bytotal IgA or total IgG concentration in each sample. Total IgA and IgGwere similarly quantitated by ELISA using plates coated with goatanti-monkey IgA or IgG (Accurate, Westbury, N.Y.), a calibrated monkeyserum standard provided by Dr. M. W. Russell of the University ofAlabama at Birmingham and the above secondary reagents.

Neutralizing antibody assays: Antibody-mediated neutralization of SIVwas measured in a CEMx174 cell-killing assay as described previously.(Langlois et al., J. Virol., 72:6950–6955 (1998), and Montefiori et al.,J. Immunol. 157:5528–5535 (1996), the teachings of which areincorporated herein in their entirety by reference). Specifically, 50 μlof cell-free virus containing 500 TCID₅₀ was added to multiple dilutionsof test serum in 100 μl of growth medium in triplicate in 96-wellculture plates. The mixtures were incubated at 37° C. for 1 hourfollowed by the addition of CEMx174 cells (5×10⁴ cells in 100 μl) toeach well. Infection led to extensive syncytium formation andvirus-induced cell killing in approximately 4–6 days in the absence ofantibodies. Neutralization was measured by staining viable cells withFinter's neutral red in poly-L-lysine-coated plates. Percent protectionwas determined by calculating the difference in absorption (A₅₄₀)between test wells (cells+serum sample+virus) and virus control wells(cells+virus), dividing this result by the difference in absorptionbetween cell control wells (cells only) and virus control wells, andmultiplying by 100. Neutralization was measured at a time whenvirus-induced cell-killing in virus control wells was greater than 70%but less than 100%. Neutralizing antibody titers ere given as thereciprocal dilution required to protect 50% of cells from virus-inducedkilling. Neutralization was measured with two stocks of SIV: 1) alaboratory-adapted stock of SIVmac251 produced in H9 cells and 2)molecularly cloned SIVmac239/nef-open produced in rhesus PBMC by using avial of the original animal challenge virus as seed stock. The formervirus is highly sensitive to neutralization whereas the latter virus isextremely difficult to neutralize in vitro.

Example 12 Cytotoxic T lymphocyte Assays

To investigate the immune response the following procedures were used:

-   (a) In vitro stimulation of PBMC: Autologous herpes    papio-transformed B lymphoblastoid cell lines (B-LCL) were infected    at an MOI of 10 pfu/cell with a recombinant vaccinia vector    expressing the SIV mac251 Gag/Pol and the SIV mac239 Env (provided    by Dr. Panicali, Therion Biologics, Cambridge Mass.). No recombinant    vaccinia viruses expressing the SIVmac239 Gag/Pol are currently    available, but the SIVmac251 and SIVmac239 molecular clones are    almost identical in Gag and Pol proteins. Vaccinia viruses    expressing the SIVmac251 Gag/Pol proteins were used to detect CTL    induced by the SIVmac239-derived vaccine. After an overnight    incubation, cells were washed, resuspended in 10 μg/ml psoralen in    RPMI with 10% FCS (R-10), and irradiated with a long wave UV light    source for 5 minutes. Following three washes, B-LCL were used as    stimulator cells at a stimulator/responder ratio of 1:10 with    2–4×10⁶ PBMC/ml. Recombinant IL-2 was added on day 4, and cultures    were fed with fresh medium containing IL-2 twice per week until    tested for CTL activity between 10 and 14 days following    restimulation. (Johnson et al., J. Virol. 71:7711–7718 (1997), the    teachings of which are incorporated herein in their entirety by    reference).

(b) ⁵¹Cr release assay: Target cells consisted of B-LCL infected withrecombinant vaccinia viruses expressing the SIVmac251 Gag, SlVmac251 Polor the SIVmac239 Env, or as a negative control, the unmodified vacciniavirus NYCBH. Vaccinia-infected targets were prepared by incubating2.5–10×10⁶ B-LCL in log-phase growth with recombinant vaccinia at 10pfu/cell for 16 hours at 37° C. Cells were labeled with 100–150 μCi ofNa₂(⁵¹CrO₄) for 60 minutes and washed 3 times with R10. Cytolyticactivity was determined in a standard ⁵¹Cr-release assay using U-bottommicrotiter plates containing 10⁴ targets per well. Lysis was generallyexamined at effector to target ratios (E:T) of 40:1, 20:1 and 10:1,although in some cases lower E:T ratios were used due to a low number ofeffector cells. To decrease background CTL activity and enhance thedetection of SIV-specific activity and enhance the detection ofSIV-specific activity, autologous unlabeled B-LCL (cold targetinhibition) at a cold:hot target ratio of 15:1 were employed. Plateswere incubated in a humidified incubator at 37° C. for either 4 or 5hours. All assays were performed in duplicate or triplicate.Supernatants (30 μl) were harvested and counted in an automatedscintillation plate reader (Wallac MicroBeta-Plus Liquid ScintillationCounter) using scintillation plates (Lumaplates). Based on examinationof SIV-specific CTL activity in over 20 negative controls studied todate, SIV-specific CTL activity of ≧5% at two different E/T ratios wasconsidered significant.

Example 13 PBMC CTL Tetramer Analysis

In the subset of vaccinated animals that express the Mamu-A*01 allele,the frequency of CD3+CD8+ cells specific for the SIV gag 11C-M epitopewas determined using MHC tetramers (100). Monkeys were typed for thepresence of the Mamu-A*01 allele as described, using Mamu-A*01-specificPCR primers followed by sequencing to confirm the presence of theMamu-A*01 allele. (Knapp et al., Tissue Antigens 50:657–661 1997), theteachings of which are incorporated herein in their entirety byreference). The gag 11C-M epitope was immunodominant in vaccinated orinfected Mamu-A*01+ animals, and thus flow cytometric analysis of thefrequency of Mamu-A*01/gag 11C-M T cells was likely to be representativeof the SIV-specific CTL response as a whole. (Egan et al., J. Virol.73:5466–5472 (1999), the teachings of which are incorporated herein intheir entirety by reference). The frequency of tetramer-binding cells inperipheral blood was analyzed using MHC tetramers consisting of theMamu-A*01 molecule complexed with the SIV gag 11C-M peptide andcomplexed with streptavidin-APC (Molecular Probes, Eugene, Oreg.)(kindly provided by Dr. John Altman, Emory University). Antibodies usedincluded FITC-conjugated anti-human CD3 (SP34, Pharmingen, San Diego,Calif.), and PerCP-conjugated anti-human CD8 (Becton Dickinson, MountainView, Calif.). Simultest reagents were used as FITC/PE isotype controls(Becton Dickenson). Analysis was performed using a FACSCalibur flowcytometer (Becton Dickenson). In general, more than 200,000 events wereacquired and analysis of tetramer staining cells was carried out on CD3⁺CD8⁺ gated lymphocytes. Concurrent negative controls consisting ofvaccinated or infected Mamu-A*01-negative animals were carried out ateach time point and gates established so as to yield less than 0.01 to0.04% tetramer-binding cells in negative controls.

Example 14 RT-PCR to Investigate RNA Viral Loads

Plasma SIV RNA levels were measured by a real time RT PCR assay,essentially as described. (Suryanarayana et al., AIDS Res. Hum.Retroviruses 14:183–189 (1998), the teachings of which are incorporatedherein in their entirety by reference). The assay has a thresholdsensitivity of 300 copy Eq/ml. Interassay variation is <25% (coefficientof variation).

Example 15 PBMC Limiting Dilution and Flow Cytometry

Cell-associated virus loads were measured by limiting dilution cultureof PBMC every month during the post-challenge time course. (Wyand etal., J. Virol. 70:3724–3733 (1996), the teachings of which areincorporated herein in their entirety by reference). Twelve 3-folddilutions of PBMC, starting with 10⁶ PBMC in the first dilution, wereprepared. Each dilution was assayed in duplicate. The PBMC wereco-cultured with a constant number of CEMx174 cells for 21 days, afterwhich the supernatant was harvested and assayed for virus-associatedp27. The titer was defined as the dilution where 50% of the wells werepositive. Therefore a titer of 1 means that 10⁶ PBMC are needed toproduce infection in one of the duplicates of the dilution and a titerof 12 means that 6 PBMC are sufficient to produce one positive CEMx174culture of the two tested.

Whole blood collected in EDTA was analyzed for lymphocyte subset CD4(OKT4a, Ortho, and/or Anti-Leu 3a, Becton Dickinson), CD8 (Anti-Leu 2a,Becton Dickinson), and CDw29 (4B4, Coulter Immunology) by a whole bloodlysis technique previously described in Wyand et al., J. Virol.70:3724–3733 (1996). Specifically, antibody (volume dependent uponantibody) was added to 100 μl of whole blood and incubated for 10minutes in the dark. Lysing solution (Becton Dickinson) was added andcells were fixed with 0.5% paraformaldehyde. Samples were analyzed onBecton Dickinson FACScan cytometer.

Example 16 Testing for Infectivity and Morphology of Mutant SIVParticles

The SIV vectors constructed were tested for particle production and lackof infectivity in a tissue culture system. Viral supernatants derivedfrom transfection were tested in infectivity assays using CEMx174 cells.A time course analysis of infections by all viruses was carried out for30 days after infection, during which the level of particle-associatedp27 was measured. Nested PCR on cellular DNA and RT-PCR on RNA frompelleted supernatants were carried out on cultures that scored negativein p27 ELISA. The results of PCR and RT-PCR were consistent with theother assays, indicating that the cell cultures were not infected (datanot shown). Particles produced from the vectors were also characterizedbiochemically (FIGS. 1A–C). As expected, a particle whose proteincomposition was similar to that of wild-type virions was assembled evenwhen the genomic RNA cannot be packaged (FIGS. 1A and B). Multiplemutations can be combined with no significant effect on particleassembly. Viral RNA was extracted from particles and quantitative RT-PCRwas subsequently performed on RNA samples. These SIV vectors, containinga total of 22 mutations affecting the function of three essential genesof SIV, produced efficient non-infectious particles containing all majorSIV proteins and no detectable viral RNA (FIG. 1C).

The different promoter efficiencies were measured by evaluating genomicviral RNA accumulation by RT-PCR in 293T transfected cells 48 hoursafter transfection (FIG. 1D). The construct pVacc2, containing the EF1apromoter, produced higher levels of RNA than construct pVacc1,containing the CMV promoter, or pMA22polyA, containing the SIV LTR, andthe increased RNA accumulation correlated with the increase in particleproduction from the transfected cells.

The morphology of the mutant particles was examined using electronmicroscopy (FIG. 2). SIV mutant particles produced from transientlytransfected cells have cores that are less electron dense than maturewild-type particles. Without be bound by theory, lack of an electrondense core could be due to the absence or incorrect positioning of theRNA and/or to less efficient precursor processing. It is possible thatthe presence of an RNA dimer is critical for achieving the correctmorphology of the particle, as the RNA might function as a scaffold inparticle assembly and maturation. (Campbell et al., J. Virol.69:6487–6497 (1995), the teachings of which are incorporated herein intheir entirety by reference).

Example 17 Generation of Mucosal and Systemic Immunity

Mucosal immunity involves some unique aspects. For example, IgA iselaborated upon a sufficient immunogenic challenge to the mucosal area.The immune system particular to the mucosal area is of great importancegiven that the infectious agent, e.g., virus, can be prevented fromentering the rest of the host, hence preventing systemic infection.Evaluation of the induction of SIV specific mucosal and systemicimmunity in primates was performed. Nine rhesus macaques were inoculatedwith SIV DNA (groups 1–3) and 3 rhesus macaques with the control plasmidpUC19 (group 4). Three different vaccination regimens were used in orderto investigate the ability of the DNA vaccine to prime differentimmunological compartments. The rationale was to compare a relativelysimple regimen of immunization to more complex regimens.

A first regimen involved intradermal DNA immunization with DNA deliveredto a region of the skin whose lymphatics drain to the iliac lymph nodes.A second regimen involved simultaneous inoculation at 1) the intradermalsite used in the first regimen, and 2) the rectal mucosa. A thirdregimen was identical to the second, except that it included, inaddition, intramuscular delivery of the DNA. The skin has the advantagethat antigen presenting cells such as dendritic cells and Langerhanscells occur at high density in the epidermis and dermis, and this may beresponsible for the relative efficiency of immunostimulation achievedthrough this route. Expression of DNA in the epidermis might be shorterlived than in muscle since epidermal cells ultimately migrate to moresuperficial layers during maturation and are sloughed off, buttransfection of subepidermal cells could also occur and some of thesemay migrate to draining lymphoid organs. DNA was deliveredintramuscularly in the third regimen in the hope of creating conditionsfor long lasting stimulation, as DNA has been found to be expressed fora considerable length of time when introduced into the skeletal muscle.(Wolff et al., Hum. Mol. Genet. 1:363–369 (1992), the teachings of whichare incorporated herein in their entirety by reference). Our rationalewas that the simplest regimen (group 1) could induce both systemicantibodies and possibly local secretory antibodies at mucosal surfacesthat are drained by iliac lymph nodes. In the more complex regimens,parenteral and mucosal immunity was simultaneously stimulated. Thevaccination schedule mimics the timetable used for hepatitis Bvaccination. Because hepatitis B is the only chronic virus for which aprotective vaccine is available and only one schedule could beinvestigated with the limited number of animals available.

Various samples were harvested during the course of the immunizationsand the following immunological assays were performed: SIV-specific IgG,IgA and neutralizing activity in the serum, SIV-specific IgA and IgG inthe rectal secretions, CTL activity in PBMC, and tetramer staining inMamu A*01 positive macaques (2 of the 9 that received the vaccine). Theresults of the immune response assays prior to live virus challenge arebriefly summarized in Table 6.

TABLE 6 Summary of immune responses to SIV DNA vaccines Antibodyresponses Tetramer Group Route SIV IgG⁷ SIV IgA⁸ CTL⁹ staining¹⁰ I (3,pVacc1) i.d. 3/3 1/3 3/3 II (3, pVacc1) i.d./R 2/3 3/3 1/3 2/2 III (3,pVacc1/2) i.d./R/i.m. 3/3 1/3 2/3 IV (3, pUC18) i.d./R/i.m. 03/ 0/3 0/3⁷IgG: serum samples 1:100–1:2560 dilution ⁸IgA: rectal secretion samples1:23–1:2179 ⁹SIV-specific CTL activity was scored positive when ≧5%, atany time after vaccination ¹⁰Tetramer staining of Mamu-A*01 positiveanimals (2) with 11/C-M Gag peptide, was considered positive when higherthan 0.05%

The most striking of all measured immune responses were the levels ofvirus-specific IgA detected in rectal secretions of animals in group 2,which received the i.d./R. regimen. SIV-specific IgG titres in serumwere weak in all groups, as has been observed previously with DNAvaccines. Virus-specific CTL activity was generally low and sporadic.

The analysis of SIV-specific IgA antibodies in rectal secretionscollected two weeks after the third vaccination is shown in Table 7.

TABLE 7 SIV specific IgA antibodies in rectal secretions on day ofchallenge SIV SIV IgA gp130 IgA IgG Specific Fold Specific Fold FoldGroup Route activity^(a) Increase^(b) activity increase increase I 19775i.d. 0.6 0.7 0.91 6.0+ nd^(c) 19796 0.65 2.2 0 nd 19831 0.89 1.3 0.322.7  5.2+ II 19777 i.d./M 1.68 23.9+ 0 nd 19786 7.06 54.1+ 28.94 87.5+ 17.55+ 19821 1.65 39.5+ 1.19 26.2+ nd III 18781 i.d./M/ 0 0 nd 18784i.m. 0.60 3.0 0.42 1.7 1.3 19856 1.23 6.6+ 0 nd IV 19783 i.d./M/ 0.693.2 0.11 1.0 1.6 19816 i.m. 0 0 nd 19845 controls 0.49 3.0 0.16 1.3 nd+indicates samples with significant levels of virus specific IgAantibodies. Significance: values of specific activity above the mean ofall preimmune samples | 3 standard deviations (99% confidence inteval)^(a)Specific activity: Anti-SIV IgA units/micrograms total IgA,signifcant at ≧1 U/μg anti-gp 130 IgA/micrograms total IgA, significantat ≧0.46 ng/μg ^(b)Fold Increase: ratio between specific activity inpost-immunization sample and specific activity in preimmune sample^(c)nd—no detectable SIV specific IgG antibodies Total IgA concentration(microgram/ml) in secretions from significant responders wererespectively: 1290 (19775), 23.5 (19777), 34.8 (19786), 3.8 (19821), and32.9 (19856).

Samples from five of nine vaccinated animals were positive at asecretion dilution of 1:23 to 1:2179. No virus-specific IgA was detectedin serum samples collected at the same time (data not shown). Thesecretions from two animals were also SIV-IgG positive. Analysis ofSIV-specific IgA content in secretions collected one month after thefirst and second vaccinations indicated that three rectal mucosal doseswere necessary to induce significant and consistent SIV-specific IgAlevels (data not shown). The data show that the administration of a DNAvaccine at the rectal mucosa can stimulate significant SIV-specific IgAresponses in primate rectal secretions. The absence of detectable SIVspecific serum IgA indicates that the IgA was locally produced. Themagnitude of the increase in SIV-specific IgA content in most of thepositive rectal samples was substantially higher than that seen thus farin any other sample analyzed in SIV-vaccinated animals or in animalsinfected with SIV.

The intramuscular administration of DNA together with rectal andintradermal inoculations appeared to negatively affect the mucosalresponses (compare fold increase in animals of groups 2 and 3 in Table7). The instant invention provides evidence that simultaneous mucosaland intramuscular DNA vaccination may not be beneficial. However, theoutcome might be different if simultaneous mucosal and systemicantigenic stimulation is provided by vaccines that are not DNA-based orare administered via different routes.

Humoral systemic virus-specific immunity was investigated by measuringSIV-specific serum IgG in an ELISA assay. As expected with DNA vaccinesSIV specific IgG responses were weak, ranging from 1:100 to 1: 2560 onthe day of challenge (Table 8). (See Hosie et al., J. Virol.72:7310–7319 (1998), and Robinson et al., Nat. Med. 5:526–534 (1999),the teachings of which are incorporated herein in their entirety byreference). Neutralization assays carried out with the same samples werenegative when SIVmac251 or SIVmac239 was used in the assay. These serumsamples had no detectable neutralizing activity against the challengevirus (i.e., molecularly cloned SIVmac239), which is consistent with thelow sensitivity of this latter virus to antibody-mediated neutralizationin and makes it uncertain that neutralizing antibodies were a componentof protection in this study. Neutralization assays were not carried outwith the rectal secretions, as detergent present in these samples madethem unsuitable for cell culture.

TABLE 8 SIV-specific serum IgG titers during DNA immunization and afterchallenge week 13 week 25 week 27 week 28 week 29 week 30 week 31 week39 week 51 4 w 16 w 2 w 1 w 2 w 3 w 4 w 12 w 21 w Group DNA route postv2^(a) post v2 post v 3 post chall post chall post chall post chall postchall post chall I 19775 i.d. N N 100(N) 100 2560  2560(443) 5120 10240(3964) 19796* i.d. N N 1280(N) 640 320 100(N) 20 20 (N) 19831 i.d. N N160(N) 100 640 2560(3827) 2560 5120 (5218) II 19777 i.d./M 100  N 640(N)2560  5120  2560(313) 5120 10240 (4064) 19786 i.d./M N N N(N) N N100(58) 80 5120 (3964) 19821* i.d./M N N 100(N) 100  20 20(N) 20 20 (N)III 19781 i.d./M/i.m. 20 N 320(N) 320 2560  2560(310) 2560 100 (1376)19784 i.d./M/i.m. 160  20 1280(N) 640 5120(1034) 20480(1589) 10240 20480(3372) 19856 i.d./M/i.m. 80 N 2560(N) 1280  5120(415) 20480(415) 51205120 (4620) IV 19783 pUVC19 N N N N N 40(107) 100 10240 (5353) 19816i.d./M/i.m. N N N N N 40(334) 80 5120 (6439) 19845 pUVC19 N N N N N100(252) 100 5120 (2612) ^(a)4 s post v 2 = 4 weeks after second dose ofvaccine *indicates animals that did not become infected after challenge.Samples collected 4 weeks after first and 2 weeks after second dose ofvaccine were SIV antidody negative. Numbers in parentheses indicateliter of neutralizing antibodies. All samples were tested inneutralization assays with SIVmac239 and SIVmac251. N = negative withboth SIVmac239 and SIVmac251.

Systemic cell-mediated immunity was investigated by measuringvirus-specific CTL activity in PBMCs. CTL responses were sporadicallypresent at different levels in different animals (Table 9).

TABLE 9 SIV specific cell-mediated immunity during DNA immunizationsWeek 27 2 post Week 4 Week 8 Week 15 Week 17 Week 21 Week 25 vacc 3 Week29 Week 30 Week 31 Week 36 DNA 4 post 8 post 6 post 8 post 12 post 16post day of 2 post 2 post 3 post 9 post Group route vacc 1 vacc 1 vacc 2vacc 2 vacc 2 vacc 2 challenge challenge challenge challenge challenge I19775 i.d. —/—/ —/—/ —/—/— —/—/— —/—/— 5/4/9 —/50/25 40/53/44 NA — —19796 ″ —/—/ 6/—/— —/—/— —/—/— —/—/— —/—/— —/—/— 9/—/— 10/—/−7 — 19831 ″10/8/— —/—/ —/—/— —/—/— —/—/— 20/18/15 7/—/12 20/21/45 21/29/23 — II19777 id/R 5/—/— —/—/ —/—/— 10/7/6 —/—/— 11/9/7 —/—/— (1049) (373) NA(121) 9/9/13 12 (005) (054) 19786 ″ NA —/—/ —/—/— —/—/— —/—/— —/—/——/—/— —/—/— —/—/— — 19821 ″ —/—/ —/—/ —/—/— —/—/— —/—/— —/—/— —/—/— (01)(007) —/—/— —/—/— — — (012) (005) (006) III 19781 Id/R/im 12/—/ —/6/1311/17/32 —/5/8 NA —/5/15 —/—/— 33/—/— —/—/— 11 19856 ″ —/6/— —/—/ —/—/——/—/— —/—/— —/—/— —/—/— —/7/30 —/—/— — 19784 ″ —/—/ —/—/ —/—/— —/—/——/—/— —/—/— —/—/— —/—/— 7/—/41 — — IV 19783 pC19id/ ND ND ND ND ND ND—/—/— —/—/10 NA R/im 19816 pC19id/ ND ND ND ND ND ND NA 5/—/46 8/—/28R/im 19845 pC19id/ ND ND ND ND ND ND —/—/— 9/—/9 9/9/13 R/im The threeresults reported at each time point for each animal are percentage ofCTI activity for the following different targets. Gag/Pol/Env, Numberindicates percentage of lysis at highest ET ratio after backgroundsubtraction. Numbers in parenthesis indicate percentage of tetramerstaining. Tetramer assays were carried out on PBMC from two Mamu-A*01positive animals (19777 and 19821) and from one Mamu-*01 negative animalND not done NA not available because of technical reasons

Animals with different genetic backgrounds respond differently to avaccine and it is possible that additional injections could haveachieved more homogeneous levels of CTL responses. One animal (19775)showed a very high level of CTL activity against env (25%) and pol (50%)when assayed two weeks after the third vaccination, indicating that thisvaccine has the potential to stimulate significant cellular responses.No clear difference in the level of SIV-specific CTL activity afterchallenge was noted between vaccinated and unvaccinated infectedmacaques.

The significant levels of IgA antibodies in rectal secretions elicitedin all three animals vaccinated i.d./R provided an opportunity for apreliminary evaluation of the role of virus-specific IgA in preventionof infection. The small size of the animal groups prevents a meaningfulstatistical analysis of the challenge results. Therefore these resultsare reported anecdotally. Investigation of larger groups of animalsimmunized via the mucosal route is necessary to elucidate the role ofvirus specific mucosal immunity in infection and disease prevention.

Animals in all groups were challenged with live virus two weeks afterthe third immunization with 5000 TCID₅₀ of cloned SIVmac239,administered to the rectal mucosa. This virus amount corresponds toapproximately 10 rectal (animal infectious dose₅₀) AID₅₀. Animals werebled weekly to assay for the presence of virus in peripheral blood andto determine whether an anamnestic response to SIV antigens wasstimulated by exposure to the virus.

Anamnestic IgG responses were observed in all the animals that werepreviously SIV-IgG positive and became infected. Seroconversion could bedocumented by an increase in SIV-specific serum IgG in control animals 3weeks after challenge (Table 8). Clear evidence of an anamnesticneutralizing antibody response was detected in serum from two animals(19831 and 19784) two to three weeks after challenge, suggesting thatpriming for neutralization epitopes was induced by the DNA vaccine. Thisanamnestic response was detected with a laboratory-adapted stock ofSIVmac251 that is highly sensitive to neutralization (Table 8). Therewas no evidence of an anamnestic neutralizing antibody response asmeasured with SIVmac239.

RT-PCR was carried out to detect RNA viral loads in serum samples fromthe day of challenge, weeks 1 to 25 after challenge (Table 10 and FIG.2), and for cell-associated virus loads measured in a limiting dilutionco-cultivation assay (data not shown).

In the infected animals, viral loads peaked two weeks post-challenge andsubsequently decreased. Average viral loads were lower for the groupvaccinated intradermally than for the control group, with differences ofapproximately 10 fold as measured by RT-PCR (FIG. 2). Two of the ninevaccinated animals (19796 of the i.d. group and 19821 of the i.d./Rgroup) remained RT-PCR negative up to week 25 post-challenge (lastavailable measurement). They also remained virus negative in a limitingdilution co-cultivation assay of their PBMC with CEMx174 cells carriedout up to 63 weeks post-challenge. PCR analysis of PBMC DNA obtainedfrom samples collected two weeks after challenge, when viremia peaked inall infected animals, was negative in the two animals that resistedchallenge (data not shown). These animals did not show IgG anamnesticresponses, possibly suggesting lack of or locally contained infection.

TABLE 10 SIV RNA viral loads post-challenge RNA copy number (× 10⁶) Dayof Group DNA route challenge week 1 week 2 week 3 week 4 week 6 week 8week 25 I 19775 i.d. < 1.3 23 8.8 0.59 0.53 0.46 0.8 19796* ″ < < < < << < < 19831 ″ < 0.17 15 8.6 1.4 0.58 0.47 1.5 II 19777 i.d./R < 0.38 205.8 9.9 3 5.8 2.4 19786 ″ < 0.06 170 49 4 4.7 3.6 0.99 19821* ″ < < < << < < < III 18781 i.d./R/i.m. < 0.23 51 4.7 15 4.1 12 6.7 19784 ″ < 1.23.8 0.16 0.28 0.19 0.34 6.8 19856 ″ < 0.14 44 6.6 1.7 2.1 1.2 4.7 IV19783 PUC19 < 0.28 3.4 0.6 12 2.6 3.3 5.8 19816 i.d./R/i.m. < 0.54 55 302.4 5.2 22 19 19845 ″ < 0.51 98 14 0.58 0.56 0.17 0.28 ″ Viral loadswere measured by quantitative RT-PCR. < indicates samples that are belowlevel of detection (threshold sensitivity: 300 copy Eq/mL) *indicatesanimals that were not infected after challenge.

PBMC FACS analysis was carried out for the T-cell immunological markersCDw29, CD4 and CD8 (Table 11). CDw29 measures a subpopulation of CD4cells (memory CD4 cells) (Morimoto et al., Selective immunomodulation:utilization of CD29/VLA molecules. Artif. Organs. 20:828–831 (1996)),and its decline has been observed as an early indicator of theimmunological decline that is correlated with subsequent diseaseprogression. (Heinkelein et al., J. Acquir. Immune Defic. Syndr.16:74–82 (1997), the teachings of which are incorporated herein in theirentirety by reference). Two consecutive measurements of this marker thatare below 10% are considered an indication of incipient immunologicaldecline. SIV-infected animals vaccinated intradermally maintained valuesof CDw29, CD4 and CD8 within the normal range for a longer period oftime than the other infected animals, while a decline affecting inparticular the CDw29 marker was evident in most of the other infectedanimals. Animals 19781 and 19784 in group 3 and 19816 in group 4 werediagnosed with an AIDS-related illness and euthanized 41 to 49 weeksafter challenge.

TABLE 11 PBMC FACS analysis CDw29 CD4 CD8 Group DNA route 0^(a) 35 47 630 15 47 63 0 35 47 63 I 19775 i.d. 16 9 10  9 27 22 23 17 15 24 27 2119796* ″ 17 18  13  18  25 29 30 37 30 39 33 38 19831 ″ 21 12  8 12  3835 29 35 30 42 43 43 II 19777 i.d./R 12 5 2 3 20 13  8  8 27 47 35 2719786 ″ 12 5 3 3 20 14 11  8 16 39 36 28 19821* ″ 14 14  11  15  22 3031 39 33 34 31 29 III 18781+ i.d./R/i.m.  9 8 4 3 13 22 17 16 21 42 4052 19784 20 9 1 2 35 22  5  7 31 37 40 21 19856 17 9 6 8 33 28 22 25 2844 23 41 IV 19783 PUC19 i/d/R/i.m.  8 12  4 4 10 35 15 20 13 29 24 2219816+ ″ 17 8 4 5 31 51 42 47 15 28 32 42 19845 ″ 15 7 9 10  29 20 27 3519 24 26 30 Numbers indicate percent age of PBMC that stains withantibodies CDw29, CD4, and CD*. Two consecutive measurements of theCDw29 marker that are below 10% are considered an indication ofimmunological decline. ^(a)numbers indicate weeks from firstimmunization. Weeks 35, 47 and 63 correspondence to respectively 8, 20and 36 weeks after challenge. *indicates animals that did not becomeinfected after challenge. +indicates animals that were diagnosed withAIDS and euthanized 9.5 months after challenge.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments on the present invention. Such equivalents are intended tobe encompassed by the following claims. All documents, patents and otherpublications cited herein are expressly incorporated by reference.

1. A nucleic acid construct which encodes non-infectious HIV particles,the construct comprising: a) at least one replacement mutation in thegag gene of a HIV genome of a HIV particle wherein the gag gene encodesthe amino acid sequence of the nucleocapsid (NC) protein, wherein thereplacement mutation produces a single amino acid substitution in atleast one position selected from the group consisting of the 5′ flankingregion (amino acids 1–14 of SEQ ID NO: 8), the 5′ Cys-His box (aminoacids 15–28 of SEQ ID NO: 8), to peptide linker region (amino acids29–35 of SEQ ID NO: 8), the 3′ Cys-His box (amino acids 36–49 of SEQ IDNO: 8) and the 3′ flanking region (amino acids 50–55 of SEQ ID NO: 8),such that the replacement mutation prevents viral RNA packaging; and b)at least one further replacement mutation in the pol gene of said HIVgenome selected from the group consisting of (i) at least onereplacement mutation in the region of the pol gene that encodes theamino acid sequence in the finger domain (amino acid 1 to 84 of SEQ IDNO: 4) of the reverse transcriptase (RT) protein, wherein thereplacement mutation produces a single amino acid substitution in thefinger domain such that a dysfunctional reverse transcriptase protein isproduced to prevent viral RNA conversion to DNA and (ii) at least onereplacement mutation in the region of the pol gene that encodes theamino acid sequence of the catalytic (amino acids 51–212 of SEQ ID NO:6) or multimerization domain (amino acids 1–50 of SEQ ID NO: 6) of theIntegrase (In) protein, wherein the replacement mutation produces asingle amino acid substitution in the catalytic or multimerizationdomain such that dysfunctional Integrase protein is produced to preventa transcribed viral DNA molecule from integrating into a host cellgenome.
 2. The construct of claim 1, wherein the mutation in the NCprotein is made in the 5′ flanking region.
 3. The construct of claim 2,wherein the mutation in the 5′ flanking region is made in at least oneposition selected from the group consisting of arginine 3, arginine 10,lysine 11 and lysine
 14. 4. The construct of claim 3, wherein the atleast one position are substituted with alanine.
 5. The construct ofclaim 1, wherein the mutation in the NC protein is made in the 5′Cys-His box.
 6. The construct of claim 5, wherein the mutation in the 5′Cys-His box is made in at least one position selected from the groupconsisting of lysine 20, histidine 23 and arginine
 26. 7. The constructof claim 6, wherein the at least one position are substituted withalanine.
 8. The construct of claim 1, wherein the mutation in the NCprotein is made in the peptide linker region.
 9. The construct of claim8, wherein the mutation in the peptide linker region is made in at leastone position selected from the group consisting of arginine 29, arginine32, lysine 33 and lysine
 34. 10. The construct of claim 9, wherein theat least one position are substituted with alanine.
 11. The construct ofclaim 1, wherein the mutation in the NC protein is made in the 3′Cys-His box.
 12. The construct of claim 11, wherein the mutation in the3′ Cys-His box is made in at least one position selected from the groupconsisting of lysine 38, lysine 41, histidine44 and lysine
 47. 13. Theconstruct of claim 12, wherein the at least one position are substitutedwith alanine.
 14. The construct of claim 1, wherein the mutation in theNC protein is made in the 3′ flanking region.
 15. The construct of claim14, wherein the mutation in the 3′ flanking region is made at positionarginine
 52. 16. The construct of claim 15, wherein the positionarginine 52 is substituted with alanine.
 17. The construct of claim 1,wherein the mutation in the RT protein is made in at least one positionselected from the group consisting of tryptophan 71, arginine 72 andarginine
 78. 18. The construct of claim 17, wherein the at least oneposition are substituted with alanine.
 19. The construct of claim 1,wherein the mutation in the In protein is made in the catalytic domain.20. The construct of claim 19, wherein the mutation in the catalyticdomain is made in at least one position selected front the groupconsisting of aspartic acid 66, aspartic acid 118 and glutamic acid 154.21. The construct of claim 20, wherein the at least one position aresubstituted with alanine.
 22. The construct of claim 1, wherein themutation in the In protein is made in the multimerization domain. 23.The construct of claim 22, wherein the mutation in the multimerizationdomain is made in at least one position selected from the groupconsisting of histidine 14, histidine 18, cysteine 42 and cysteine 45.24. The construct of claim 23, wherein the at least one position aresubstituted with alanine.
 25. The construct of claim 1, wherein theconstruct comprises a cluster of mutations, wherein the cluster ofmutations is made in a region selected from the group consisting of the5′ flanking region of the NC protein, the 5′ Cys-His box of the NCprotein, the peptide linker region of the NC protein, the 3′ Cys-His boxof the NC protein, the 3′ flanking region of the NC protein, a β3–β4loop of the RT protein, the catalytic domain of the In protein and themultimerization domain of the In protein.
 26. The construct of claim 1,wherein the construct comprises mutations in the NC protein at positionslysine 14, lysine 20, arginine 26, arginine 29, arginine 32, lysine 33,lysine 34, lysine 38, lysine 41 and lysine 47, mutations in the RTprotein at positions tryptophan 71, arginine 72 and arginine 78, andmutations in the In protein at positions histidine 14, histidine 18,cysteine 42, cysteine 45, aspartic acid 66, aspartic acid 118 andglutamic acid
 154. 27. The construct of claim 26, wherein the at leastone position are substituted with alanine.
 28. The construct of claim 1,further comprising a cytomegalovirus (CMV) promoter replacing a HIV 5′long terminal repeat (LTR) sequence.
 29. The construct of claim 28,wherein the cytomegalovirus (CMV) promoter replaces the HIV 5′ longterminal repeat (LTR) sequence from nucleotide 1 to nucleotide 636 ofSEQ ID NO:
 1. 30. The construct of claim 1, further comprising anelongation factor 1a (EF1a) promoter replacing a HIV 5′ long terminalrepeat (LTR) sequence.
 31. The construct of claim 30, wherein theelongation factor 1a (EF1a) promoter replaces the HIV 5′ long terminalrepeat (LTR) sequence from nucleotide 1 to nucleotide 636 of SEQ IDNO:
 1. 32. The construct of claim 1, further comprising an SV40polyadenylation signal replacing a HIV 3′ long terminal repeat sequence.33. The construct of claim 32, wherein the SV40 polyadenylation signalreplaces the HIV 3′ long terminal repeat (LTR) sequence from nucleotide8902 to the end of SEQ ID NO:
 1. 34. The construct of claim 1, whereinthe construct further comprises a selectable marker gene.
 35. Theconstruct of claim 34, wherein said selectable marker gene encodes aselectable marker which is selected from the group consisting ofneomycin resistance, hygromycin resistance and dihydrofolate reductase.36. A mammalian culture cell line, transfected with the construct ofclaim 1, which produces mutant virions.
 37. The cell line of claim 36,wherein the construct is stably integrated in the genome of the cellline, and the cell line stably produces the non-infectious HIVparticles.
 38. An immunogenic composition to reduce viral loadcomprising the nucleic acid construct of claim
 1. 39. An immunogeniccomposition to reduce viral load comprising the nucleic acid constructof claim
 26. 40. An immunogenic composition to reduce viral loadcomprising the nucleic acid construct of claim 1, where at least one gagcodon and at least one pol codon are optimized according to the codonusage in man.
 41. An immunogenic composition to reduce viral loadcomprising the nucleic acid construct of claim 26, where at least onegag codon and at least one pol codon are optimized according to thecodon usage in man.
 42. A nucleic acid construct which encodesnon-infectious HIV particles, the construct comprising: a) at least onemutation in the gag gene of a HIV genome of a HIV particle wherein thegag gene encodes the amino acid sequence of the nucleocapsid (NC)protein, wherein the mutation is made in the 5′ flanking region (aminoacids 1–14 of SEQ ID NO: 8), such that the mutation prevents viral RNApackaging; and b) at least one further mutation in the pol gene of saidHIV genome selected from the group consisting of (i) at least onemutation in the region of the pol gene that encodes the amino acidsequence in the finger domain (amino acid 1 to 84 of SEQ ID NO: 4) ofthe reverse transcriptase (RT) protein, such that a dysfunctionalreverse transcriptase protein is produced to prevent viral RNAconversion to DNA and (ii) at least one mutation in the region of thepol gene that encodes the amino acid sequence of the catalytic (aminoacids 51–212 of SEQ ID NO: 6) or multimerization domain (amino acids1–50 of SEQ ID NO: 6) of the Integrase (In) protein, such thatdysfunctional Integrase protein is produced to prevent a transcribedviral DNA molecule from integrating into a host cell genome.
 43. Theconstruct of claim 42, wherein the mutation in the 5′ flanking region ismade in at least one position selected from the group consisting ofarginine 3, arginine 10, lysine 11 and lysine
 14. 44. The construct ofclaim 43, wherein the at least one position are substituted withalanine.
 45. A nucleic acid construct which encodes non-infectious HIVparticles, the construct comprising: a) at least one mutation in the gaggene of a HIV genome of a HIV particle wherein the gag gene encodes theamino acid sequence of the nucleocapsid (NC) protein, wherein themutation is made in the 5′ Cys-His box in at least one position selectedfrom the group consisting of lysine 20, histidine 23 and arginine 26,such that the mutation prevents viral RNA packaging; and b) at least onefurther mutation in the pol gene of said HIV genome selected from thegroup consisting of i) at least one mutation in the region of the polgene that encodes the amino acid sequence in the finger domain (aminoacid 1 to 84 of SEQ ID NO: 4) of the reverse transcriptase (RT) proton,such that a dysfunctional reverse transcriptase protein is produced toprevent viral RNA conversion to DNA and (ii) at least one mutation inthe region of the pol gene that encodes the amino acid sequence of thecatalytic (amino acids 51–212 of SEQ ID NO: 6) or multimerization domain(amino acids 1–50 of SEQ ED NO: 6) of the Integrase (In) protein, suchthat dysfunctional Integrase protein is produced to prevent atranscribed viral DNA molecule from integrating into a host cell genome.46. The construct of claim 45, wherein the at least one position aresubstituted with alanine.
 47. A nucleic acid construct which encodesnon-infections HIV particles, the construct comprising: a) at least onemutation in the gag gene of a HIV genome of a HIV particle wherein thegag gene encodes the amino acid sequence of the nucleocapsid (NC)protein, wherein the mutation is made in the peptide linker region(amino acids 29–35 of SEQ ID NO: 8), such that the mutation preventsviral RNA packaging; and b) at least one further mutation in the polgene of said HIV genome selected from the group consisting of (i) atleast one mutation in the region of the pol gene that encodes the aminoacid sequence in the finger domain (amino acid 1 to 84 of SEQ ID NO: 4)of the reverse transcriptase (RT) protein, such that a dysfunctionalreverse transcriptase protein is produced to prevent viral RNAconversion to DNA and (ii) at least one mutation in the region of thepol gene that encodes the amino acid sequence of the catalytic (aminoacids 51–212 of SEQ ID NO: 6) or multimerization domain (amino acids1–50 of SEQ ID NO: 6) of the Integrase (In) protein, such thatdysfunctional Integrase protein is produced to prevent a transcribedviral DNA molecule from integrating into a host cell genome.
 48. Theconstruct of claim 47, wherein the mutation in the peptide linker regionis made in at least one position selected from the group consisting ofarginine 29, arginine 32, lysine 33 and lysine
 34. 49. The construct ofclaim 48, wherein the at least one position are substituted withalanine.
 50. A nucleic acid construct which encodes non-infectious HIVparticles, the construct comprising: a) at least one mutation in the gaggene of a HIV genome of a HIV particle wherein the gag gene encodes theamino acid sequence of the nucleocapsid (NC) protein, wherein themutation is made in the 3′ Cys-His box (amino acids 36–49 of SEQ ID NO:8), such that the mutation prevents viral RNA packaging; and b) at leastone further mutation in the pol gene of said HIV genome selected fromthe group consisting of (i) at least one mutation in the region of thepol gene that encodes the amino acid sequence in the finger domain(amino acid 1 to 84 of SEQ ID NO: 4) of the reverse transcriptase (RT)protein, such that a dysfunctional reverse transcriptase protein isproduced to prevent viral RNA conversion to DNA and (ii) at least onemutation in the region of the pol gene that encodes the amino acidsequence of the catalytic (amino acids 51–212 of SEQ ID NO: 6) ormultimerization domain (amino acids 1–50 of SEQ ID NO: 6) of theIntegrase (In) protein, such that dysfunctional Integrase protein isproduced to prevent a transcribed viral DNA molecule from integratinginto a host cell genome.
 51. The construct of claim 50, wherein themutation in the 3′ Cys-His box is made in at least one position selectedfrom the group consisting of lysine 38, lysine 41, histidine 44 andlysine
 47. 52. The construct of claim 51, wherein the at least oneposition are substituted with alanine.
 53. A nucleic acid constructwhich encodes non-infectious HIV particles, the construct comprising: a)at least one mutation in the gag gene of a HIV genome of a HIV particlewherein the gag gene encodes the amino acid sequence of the nucleocapsid(NC) protein, wherein the mutation is made in the 3′ flanking region(amino acids 50–55 of SEQ ID NO: 8), such that the mutation preventsviral RNA packaging; and b) at least one further mutation in the polgene of said HIV genome selected from the group consisting of (i) atleast one mutation in the region of the pol gene that encodes the aminoacid sequence in the finger domain (amino acid 1 to 84 of SEQ ID NO: 4)of the reverse transcriptase (RT) protein, such that a dysfunctionalreverse transcriptase protein is produced to prevent viral RNAconversion to DNA and (ii) at least one mutation in the region of thepol gene that encodes the amino acid sequence of the catalytic (aminoacids 51–212 of SEQ ID NO: 6) or multimerization domain (amino acids1–50 of SEQ ED NO: 6) of the Integrase (In) protein, such thatdysfunctional Integrase protein is produced to prevent a transcribedviral DNA molecule from integrating into a host cell genome.
 54. Theconstruct of claim 53, wherein the mutation in the 3′ flanking region ismade at position arginine
 52. 55. The construct of claim 54, wherein theat least one position are substituted with alanine.
 56. A nucleic acidconstruct which encodes non-infectious HIV particles, the constructcomprising: a) at least one mutation in the gag gene of a HIV genome ofa HIV particle wherein the gag gene encodes the amino acid sequence ofthe nucleocapsid (NC) protein, wherein the mutation is made in at leastone position selected from the group consisting of the 5′ flankingregion (amino acids 1–14 of SEQ ID NO: 8), the 5′ Cys-His box (aminoacids 15–28 of SEQ ID NO: 8), the peptide linker region (amino acids29–35 of SEQ ID NO: 8), the 3′ Cys-His box (amino acids 36–49 of SEQ IDNO: 8) and the 3′ flanking region (amino acids 50–55 of SEQ ID NO: 8),such that the mutation prevents viral RNA packaging; and b) at least onereplacement mutation in the pol gene of said HIV genome selected fromthe group consisting of (i) at least one replacement mutation in theregion of the pol gene that encodes the amino acid sequence in thefinger domain (amino acid 1 to 84 of SEQ ID NO: 4) of the reversetranscriptase (RT) protein, wherein the replacement mutation in the RTprotein produces a single amino acid substitution in at least oneposition selected from the group consisting of tryptophan 71, arginine72 and arginine 78, and wherein the amino acid positions are substitutedwith alanine, such that a dysfunctional reverse transcriptase protein isproduced to prevent viral RNA conversion to DNA and (ii) at least onereplacement mutation in the region of the pol gene that encodes theamino acid sequence of the catalytic (amino acids 51–212 of SEQ ID NO:6) or multimerization domain (amino acids 1–50 of SEQ ID NO: 6) of theIntegrase (In) protein, wherein the replacement mutation produces asingle amino acid substitution in the catalytic or multimerizationdomain such that dysfunctional Integrase protein is produced to preventa transcribed viral DNA molecule from integrating into a host cellgenome.
 57. A nucleic acid construct which encodes non-infectious HIVparticles, the construct comprising: a) at least one mutation in the gaggene of a HIV genome of a HIV particle wherein the gag gene encodes theamino acid sequence of the nucleocapsid (NC) protein, wherein themutation is made in at least one position selected from the groupconsisting of the 5′ flanking region (amino acids 1–14 of SEQ ID NO: 8),the 5′ Cys-His box (amino acids 15–28 of SEQ ID NO: 8), the peptidelinker region (amino acids 29–35 of SEQ ID NO: 8), the 3′ Cys-His box(amino acids 36–49 of SEQ ID NO: 8) and the 3′ flanking region (aminoacids 50–55 of SEQ ID NO: 8), such that the mutation prevents viral RNApackaging; and b) at least one replacement mutation in the pol gene ofsaid HIV genome selected from the group consisting of (i) at least onereplacement mutation in the region of the pol gene that encodes theamino acid sequence in the finger domain (amino acid 1 to 84 of SEQ IDNO: 4) of the reverse transcriptase (RT) protein, wherein thereplacement mutation produces a single amino acid substitution in thefinger domain such that a dysfunctional reverse transcriptase protein isproduced to prevent viral RNA conversion to DNA and (ii) at least onereplacement mutation in the region of the pol gene that encodes theamino acid sequence of the catalytic domain (amino acids 51–212 of SEQID NO: 6) of the Integrase (In) protein, wherein the replacementmutation in the catalytic domain produces a single amino acidsubstitution in at least one position selected from the group consistingof aspartic acid 66, aspartic acid 118 and glutamic acid 154, andwherein the amino acid positions are substituted with alanine, such thatdysfunctional Integrase protein is produced to prevent a transcribedviral DNA molecule from integrating into a host cell genome.
 58. Anucleic acid construct which encodes non-infectious HIV particles, theconstruct comprising: a) at least one mutation in the gag gene of a HIVgenome of a HIV particle wherein the gag gene encodes the amino acidsequence of the nucleocapsid (NC) protein, wherein the mutation is madein at least one position selected from the group consisting of the 5′flanking region (amino acids 1–14 of SEQ ID NO: 8), the 5′ Cys-His box(amino acids 15–28 of SEQ ID NO: 8), the peptide linker region (aminoacids 29–35 of SEQ ID NO: 8), the 3′ Cys-His box (amino acids 36–49 ofSEQ ID NO: 8) and the 3′ flanking region (amino acids 50–55 of SEQ IDNO: 8), such that the mutation prevents viral RNA packaging; and b) atleast one replacement mutation in the pol gene of said HIV genomeselected from the group consisting of (i) at least one replacementmutation in the region of the pol gene that encodes the amino acidsequence in the finger domain (amino acid 1 to 84 of SEQ ID NO: 4) ofthe reverse transcriptase (RT) protein, wherein the replacement mutationproduces a single amino acid substitution in the finger domain such thata dysfunctional reverse transcriptase protein is produced to preventviral RNA conversion to DNA and (ii) at least one replacement mutationin the region of the pol gene that encodes the amino acid sequence ofthe multimerization domain (amino acids 1–50 of SEQ ID NO: 6) of theIntegrase (In) protein, wherein the replacement mutation in themultimerization domain produces a single amino acid substitution in atleast one position selected from the group consisting of histidine 14,histidine 18, cysteine 42 and cysteine 45, and wherein the amino acidpositions are substituted with alanine, such that dysfunctionalIntegrase protein is produced to prevent a transcribed viral DNAmolecule from integrating into a host cell genome.
 59. A nucleic acidconstruct which encodes non-infectious HIV particles, the constructcomprising: a) at least one mutation in the gag gene of a HIV genome ofa HIV particle wherein the gag gene encodes the amino acid sequence ofthe nucleocapsid (NC) protein, wherein the mutation is made in at leastone position selected from the group consisting of the 5′ flankingregion (amino acids 1–14 of SEQ ID NO: 8), the 5′ Cys-His box (aminoacids 15–28 of SEQ ID NO: 8), the peptide linker region (amino acids29–35 of SEQ ID NO: 8), the 3′ Cys-His box (amino acids 36–49 of SEQ IDNO: 8) and the 3′ flanking region (amino acids 50–55 of SEQ ID NO: 8),wherein the mutations in the NC protein are at positions lysine 14,lysine 20, arginine 26, arginine 29, arginine 32, lysine 33, lysine 34,lysine 38, lysine 41 and lysine 47, such that the mutations preventsviral RNA packaging; and b) at least one further mutation in the polgene of said HIV genome selected from the group consisting of (i) atleast one mutation in the region of the pol gene that encodes the aminoacid sequence in the finger domain (amino acid 1 to 84 of SEQ ID NO: 4)of the reverse transcriptase (RT) protein, wherein the mutations in theRT protein are at positions tryptophan 71, arginine 72 and arginine 78,such that a dysfunctional RT protein is produced to prevent viral RNAconversion to DNA and (ii) at least one mutation in the region of thepol gene that encodes the amino acid sequence of the catalytic (aminoacids 51–212 of SEQ ID NO: 6) or multimerization domain (amino acids1–50 of SEQ ID NO: 6) of the Integrase (In) protein, wherein themutations in the In protein at positions histidine 14, histidine 18,cysteine 42, cysteine 45, aspartic acid 66, aspartic acid 118 andglutamic acid 154, such that dysfunctional In protein is produced toprevent a transcribed viral DNA molecule from integrating into a hostcell genome.
 60. The construct of claim 58, wherein the at least oneposition are substituted with alanine.
 61. The nucleic acid construct ofclaim 1, wherein the construct further comprises a cytomegalovirus (CMV)promoter replacing a HIV 5′ long terminal repeat (LTR) sequence.
 62. Theconstruct of claim 61, wherein the cytomegalovirus (CMV) promoterreplaces the HIV 5′ long terminal repeat (LTR) sequence from nucleotide1 to nucleotide 636 of SEQ ID NO:
 1. 63. An immunogenic composition toreduce viral load comprising the nucleic acid construct of claim 59.